MX2007004892A - Orthogonal translation components for the in vivo incorporation of unnatural amino acids. - Google Patents

Abstract

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins produced in eubacterial host cells such as <i>E.coli,</i>or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Description

ORTHOGONAL TRANSLATION COMPONENTS FOR THE IN VIVO INCORPORATION OF NON-NATURAL AMINO ACIDS FIELD OF THE INVENTION The present invention is concerned with the field of translation biochemistry. The invention is concerned with compositions and methods for making and using orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases and pairs thereof, which incorporate non-natural amino acids into proteins. The invention is also concerned with methods for producing proteins in cells using such pairs and proteins made by the methods.

BACKGROUND OF THE INVENTION The study of the structure and function of the protein has historically depended on the reaction properties and chemistries that are available using the reactive groups of amino acids that occur stably in nature. Unfortunately, each known organism, from bacteria to humans, codes for the same twenty common amino acids (with the rare exceptions of selenocysteine (see for example A. Bock et al., (1991), Molecular Microbiology 5: 515-20) and pyrrolysin ( see for example G. Srinivasan, et al., (2002), Science 296: 1459-62). This limited selection of groups R has restricted the study of protein structure and function, where the studies are confined by the chemical properties of amino acids that occur stably in nature, for example, unnatural amino acids limit the ability to make protein modifications highly targeted to the exclusion of all other amino acids in a protein. Additionally, natural amino acids are limited in their functional activities, for example fluorescence, metal chelation, redox potential, photoenchylation, etc. Most of the modification reactions currently used in the art for selective protein modification involve covalent bond formation between nucleophilic and electrophilic reaction partners that point to nucleophilic residues that occur stably in nature in the amino acid side chains of protein, for example, the reaction of α-halo ketones with histidine or cysteine side chains. The selectivity in these cases is determined by the number and accessibility of the nucleophile residues in the protein. Unfortunately, proteins that occur stably in nature often contain poorly placed (eg, inaccessible) reaction sites or multiple reaction targets (eg, lysine, histidine and cysteine residues), resulting in efficient selectivity in the Modification reactions, making the protein modification highly targeted by reagents difficult nucleophilic / electrophilic. In addition, modification sites are commonly limited to the nucleophilic side chains that occur stably in the nature of lysine, histidine or cysteine. Modification in other sites is difficult or impossible. What is needed in the art are new strategies for the incorporation of non-natural amino acids into proteins for the purpose of modifying and studying the structure and function of protein, where non-natural amino acids have novel properties, for example biological properties, not found in the amino acids that occur in a stable way in nature. There is considerable need in the art for the creation of new strategies for protein modification reactions that modify proteins in a highly selective manner and also, modify proteins under physiological conditions. What is needed in the art are new methods for producing protein modifications, where the modifications are highly specific, for example modifications where none of the amino acids that are stably present in nature are subject to cross reactions or side reactions . New chemistries for highly specific protein modification strategies can find a wide variety of applications in the study of protein structure and function.

One strategy to overcome these limitations is to expand the genetic code and add amino acids that have physical, chemical or biological properties distinctive to the biological repertoire. This procedure has proven to be feasible using orthogonal tRNAs and corresponding new orthogonal aminoacyl-tRNA synthetases to add non-natural amino acids to proteins using the in vivo protein biosynthetic machinery of a host cell, eg the eubacterium Escherichia coli (E. coli), yeast or amine cells. This procedure is described in several sources, for example Wang et al., (2001), Science 292: 498-500; Chin et al., (2002) Journal of the American Chemical Society 124: 9026-9027; Chin and Schultz, (2002), ChemiBioChem 11: 1135-1137; Chin et al., (2002), PNAS United States of America 99: 11020-11024; and Wang and Shultz, (2002), Chem. Comm. , 1-10. See also international publications WO 2002/080675, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415, filed July 7, 2004; WO 2005/007870, filed July 7, 2004; and WO 2005/007624, filed July 7, 2004. There is a need in the art for the development of orthogonal translation components that incorporate non-natural amino acids to proteins, where non-natural amino acids can be incorporated in a defined position and where non-natural amino acids impart new biological properties to the proteins in which they are incorporated. There is also a need to develop orthogonal translation components that incorporate non-natural amino acids with new chemical properties that allow the amino acid to serve as a target for specific modification to the exclusion of cross reactions or side reactions with other sites in the proteins. There is also a particular need for protein expression systems that have the ability to produce proteins containing unnatural amino acids in significant amounts that allow their use in therapeutic applications and biomedical research. The invention described herein meets these and other needs, as will be apparent from the review of the following disclosure.

BRIEF DESCRIPTION OF THE INVENTION The invention provides composition and methods for incorporating unnatural amino acids into a growing polypeptide chain in response to a selector codon, for example an amber retention codon, in vivo (e.g., in a host cell). These compositions include orthogonal t-RNA pairs (O-tRNA) and orthogonal aminoacyl-tRNA synthetases (O-RS) that do not interact with the translation machinery of the host cell. That is, the O-tRNA is not loaded (or not loaded to a significant level) with an amino acid (natural or unnatural) by an endogenous host cell aminoacyl-tRNA synthetase. Similarly, the O-RSs provided by the invention do not load any endogenous tRNA with an amino acid (natural or unnatural) at a significant level or in some cases detectable level. These new compositions allow the production of large amounts of proteins that have non-natural amino acids incorporated translationally. Depending on the chemical properties of the non-natural amino acid that is incorporated, these proteins find a wide variety of uses, including, for example, as therapeutic and in biomedical research. In some aspects, the invention provides translation systems. These systems comprise a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA) and an unnatural amino acid, wherein the first O-RS preferably aminoacylates the first O-tRNA with the first amino acid not natural . The first non-natural amino acid can be selected from p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin-alanine, 7-hydroxy-coumarin -alanine, o-nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, m-cyano-L-phenylalanine, p-cyano-L-phenylalanine, biphenylalanine, 3-amino -L-tyrosine, bipyridylalanine, p- (2- amino-1-hydroxyethyl) -L-phenylalanine and p-isopropylthiocarbonyl-L-phenylalanine. Translation systems can use components derived from a variety of sources. In one embodiment, the first O-RS is derived from an aminoacyl-tRNA synthetase from Methanococcus jannaschii, for example, a tyrosyl-tRNA synthetase from wild-type Methanococcus jannaschii. In other embodiments, O-RS is derived from an aminoacyl-tRNA synthetase from E. coli, for example, a wild-type E. coli leucyl-tRNA synthetase. The O-RS used in the system may comprise an amino acid sequence selected from SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57, 59-63 and conservative variants of those sequences. In some embodiments, the O-tRNA is an amber suppressor t-RNA. In some embodiments, the O-tRNA comprises or is encoded by SEQ ID NO: 1 or 2. In some aspects, the translation system further comprises a nucleic acid encoding a protein of interest, wherein the nucleic acid has at least a selector codon that is recognized by the O-tRNA. In some aspects, the translation system incorporates a second orthogonal pair (that is, a second O-RS and a second O-tRNA) that uses a second unnatural amino acid, such that the system is now capable of incorporating minus two different non-natural amino acids at different sites selected on a polypeptide. In this double system, the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid and the second O-tRNA recognizes a selector codon that is different from the selector codon recognized by the first O-tRNA. In some embodiments, the translation system resides in a host cell (and includes the host cell). The host cell used is not particularly limited, as long as the O-RS and the O-tRNA retain their orthogonality in their host cell environment. The host cell may be a eubacterial cell, such as an E. coli cell or a yeast cell, such as Saccharomyces cerevi siae. The host cell may comprise one or more polynucleotides encoding components of the translation system, in which the O-RS or tRNA is included. In some embodiments, the polynucleotide encoding the O-RS comprises a nucleotide sequence of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 or 58. The invention also provides methods for producing proteins having one or more non-natural amino acids at selected positions. These methods use the translation systems described above. In general, these methods start with the step of providing a translation system comprising: (i) a first amino acid not natural selected from p-ethylthiocarbonyl-L-phenylalanma, p- (3-oxobutanoyl) -L-phenylalanine, 1,5-dans? l-alanma, 7-ammo-coumarm-alanma, 7-hydrox? -coumarm- alanma, o-nitrobenzyl-serine, 0- (2-n? trobenc? l) -L-tyrosma, p-carboxymethyl-L-phenylalanm, m-cyano-L-phenylalanine, p-cyano-L-phenylalanine, biphenylalanine, 3 -ammo-L-tyrosma, bipyridilalanma, p- (2-ammo-1-hydrox? Et? L) -L-phenylalanine and p-isopropylthiocarbonyl-L-phenylalanine; (n) a first ammoacyl-tRNA orthogonal synthetase (O-RS); (ni) a first orthogonal tRNA (O-tRNA), wherein the O-RS ammoacyl preferably the O-tRNA with the unnatural amino acid and (v) a nucleic acid encoding the protein, wherein the nucleic acid comprises minus a selector codon that is recognized by the first O-tRNA. The method then incorporates the unnatural amino acid at the selected position in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising the unnatural amino acid at the selected position. These methods can be widely applied using a variety of reagents and steps. In some embodiments, a polynucleotide encoding O-RS is provided. In some embodiments, an O-RS derived from an ammoacyl-tRNA smtetase from Methanococcus annaschii, for example a tyrosyl-tRNA smtetase Methanococcus jannaschii wild type, is provided. In other modalities, an O-RS is provided derived from an ammoacyl-tRNA smtetase from E. coli, for example, an O-RS derived from a wild-type E. coll leucyl-tRNA synthetase is provided. In some embodiments, the provisioning step includes providing an O-RS comprising an amino acid sequence selected from SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57, 59-63 and conservative variants thereof In some embodiments of these methods, the step of providing a translation system comprises mutation of a cavity of amino acid linkage of a wild-type ammoacyl-TARN synthetase by site-directed mutagenesis and selecting an O-RS that preferentially ammoacylates O-tRNA with the unnatural amino acid. The selection step may comprise positively selecting and negatively screening the O-RS from a cluster of ammoacyl-tRNA smtetase molecules resulting next from site-directed mutagenesis. In some embodiments, the provisioning step provides a polmucleotide encoding the O-tRNA, for example, an O-tRNA that is an amber suppressor tRNA or an O-tRNA that comprises or is encoded by a polynucleotide of SEQ ID NO: 1 or 2. In these methods, the provisioning step can also provide a nucleic acid comprising a selector codon both which is used by the translation system. These methods can also be modified to incorporate more than one non-natural amino acid into a protein. In these methods, a second orthogonal translation system is employed in conjunction with the first translation system, wherein the second system has different amino acid and codon selector specificities. For example, the provisioning step may include providing a second O-RS and a second O-tRNA, wherein the second O-RS preferably ammoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid. and wherein the second O-tRNA recognizes a selector codon in the nucleic acid that is different from the selector codon recognized by the first O-tRNA. Methods for producing a protein with an unnatural amino acid can also be carried out in the context of a host cell. In these cases, a host cell is provided, wherein the host cell comprises the non-natural amino acid, the O-RS, the O-tRNA and the nucleic acid and wherein the culture of the host cell results in the incorporation of the amino acid not natural In some embodiments, the provisioning step comprises providing a eubacterial host cell (e.g., E. coli) or a yeast host cell. In some embodiments, the provisioning step includes providing a host cell that contains a polynucleotide that encodes O-RS. For example, the polypeptide encoding the O-RS may comprise a nucleotide sequence of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 or 58. In some embodiments, the step of providing a translation system is carried out by providing a cellular extract. In some aspects, the invention provides translation systems, wherein the systems are for the incorporation of 3-nitro-L-tyrosine or p-nitro-L-phenylalanine. These systems comprise a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA) and the non-natural amino acid, wherein the first O-RS preferably aminoacylates the first O-tRNA with the first amino acid not natural with an efficiency that is at least 50% of the efficiency observed for a translation system comprising that same non-natural amino acid, the O-tRNA and an O-RS comprising an amino acid sequence selected from SEQ ID NOs: 7 -10. The translation system can use components derived from a variety of sources. In some embodiments, the first O-RS is derived from an aminoacyl-tRNA synthetase from Methanococcus jannaschii, for example a tyrosyl-tRNA synthetase from wild-type Methanococcus jannaschii. The O-RS used in the system may comprise an amino acid sequence selected from SEQ ID NOs: 7-10 and conservative variants of those sequences. In some modalities, the O-tRNA is an amber suppressor tARN. In some modalities, the O-tRNA comprises or is encoded by SEQ ID NO: 1. In some aspects, the translation system further comprises a nucleic acid encoding a protein of interest wherein the nucleic acid has at least one selector codon that is recognized by the t-RNA In some aspects, the translation system incorporates a second orthogonal pair (that is, a second O-RS and a second O-tRNA) that uses a second non-natural amino acid, such that the system is now capable of incorporating minus two different unnatural amino acids at different selected sites in a polypeptide. In this double system, the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid and the second O-tRNA recognizes a selector codon that is different from the selector codon recognized by the first O-tRNA. In some embodiments, the translation system resides in a host cell (and includes the host cell). The host cell used is not particularly limited, as long as O-RS and O-tRNA retain their orthogonality in their host cell environment. The host cell can be a eubacterial cell, such as E. coli. The host cell may comprise one or more polynucleotides encoding components of the translation system, in which the O-RS or O-tRNA is included. In some embodiments, the polynucleotide encoding the O-RS comprises a nucleotide sequence of SEQ ID NO: 11. The invention provides methods for producing proteins having one or more non-natural amino acids at selected positions. These methods use the translation systems described above. In general, these methods start with the step of providing a host cell comprising a translation system comprising: (i) a first unnatural amino acid that is 3-nitro-L-tyrosine or p-nitro-L-phenylalanine; (ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) a first orthogonal tRNA (O-tRNA), wherein the O-RS preferably inactivates the O-tRNA with the unnatural amino acid with an efficiency that is at least 50% of the efficiency observed for the host cell that comprises the non-natural amino acid, the O-tRNA and an O-RS comprising an amino acid sequence selected from SEQ ID NOs: 7-10; and (iv) a nucleic acid encoding the protein, wherein the nucleic acid comprises at least one selector codon that is recognized by the O-tRNA. Then the host cell is cultured and the unnatural amino acid is incorporated in the selected position in the protein during the translation of the protein in response to the selector codon, where the position selected in the protein corresponds to the position of the selector codon in the acid nucleic acid, thereby producing the protein comprising the unnatural amino acid in the selected position.

These methods can be applied widely using a variety of reagents and stages. In some embodiments, a polynucleotide encoding O-RS is provided. In some embodiments, an O-RS derived from an aminoacyl-tRNA synthetase from Methanococcus jannaschii is provided, for example a tyrosyl-tRNA synthetase from Methanococcus jannaschi i wild type can be provided. In some embodiments, the provisioning step includes providing a 0-RS comprising an amino acid sequence selected from SEQ ID Nos: 7-10 and conservative variants thereof. In some embodiments of these methods, the step of providing a translation system comprises mutation of an amino acid linkage cavity of a wild-type aminoacyl-tRNA N synthetase by site-directed mutagenesis and selecting a resulting O-RS which preferably aminoacyl O- tRNA with the non-natural amino acid. The selection step may comprise positively screening and negatively screening for the O-RS of a cluster of aminoacyl-tRNA synthetase molecules resulting next from site-directed mutagenesis. In some embodiments, the provisioning step provides a polynucleotide encoding 0-tRNA, for example an O-tRNA that is an amber suppressor tRNA or an O-tRNA that comprises or is encoded by a polynucleotide of SEQ ID NO: 1. In these methods the provisioning step can also provide a nucleic acid comprising a codon Amber selector that is used by the translation system. These methods can also be modified to incorporate more than one unnatural amino acid into a protein. In these methods, a second orthogonal translation system is employed in conjunction with the first translation system, wherein the second system has different amino acid and codon selector specificities. For example, the provisioning step may include providing a second O-RS and a second O-tRNA, wherein the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid. and wherein the second O-tRNA recognizes a selector codon in the nucleic acid that is different from the selector codon recognized by the first O-tRNA. Methods for producing a protein with an unnatural amino acid are carried out in the context of a host cell. In these embodiments, the host cell comprises the non-natural amino acid, the O-RS, the O-tRNA and the nucleic acid and wherein the culture of the host cell results in the incorporation of the non-natural amino acid. In some embodiments, the provisioning step comprises providing a eubacterial host cell (e.g., E. coli). In some embodiments, the provisioning step includes providing a host cell that contains a polynucleotide that encodes O-RS. For example, the polynucleotide that encoding the O-RS may comprise a nucleotide sequence of SEQ ID NO: 11. In some embodiments, the step of providing a translation system is carried out by providing a cellular extract. The invention also provides a variety of compositions, in which nucleic acids and proteins are included. The nature of the composition is particularly limited, unlike the composition comprising the specified nucleic acid or protein. The compositions of the invention may comprise any number of additional components of any nature. For example, the invention provides compositions comprising O-RS polypeptides, wherein the polypeptides comprise SEQ ID NO: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50-55, 57, 59-63, or a conservative variant thereof, wherein the conservative variant polypeptide aminoacylates an orthogonal tRNA (O-tRNA) cognate with a non-natural amino acid with an efficiency that is at least 50% of the efficiency observed by a translation system comprising the O-tRNA, the unnatural amino acid and an aminoacyl-tRNA synthetase comprising an amino acid sequence selected from SEQ ID NOs : 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59 -63. The invention also provides polynucleotides that encode any of these polypeptides previous In some embodiments, these polynucleotides may comprise SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 and 58. In some embodiments, the polypeptides are in a cell. The invention also provides polynucleotide compositions comprising a nucleotide sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 or 58. In some embodiments, the invention provides vectors comprising the polynucleotides, for example expression vectors. In some embodiments, the invention provides cells comprising a vector described above.

DEFINITIONS Before describing the invention in detail, it will be understood that the present invention is not limited to particular biological systems, which may vary of course. It will also be understood that the terminology herein is for the purpose of describing particular modalities only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, the reference to "a cell" includes combinations of two or more cells; the reference to "a polynucleotide "includes, of course, many copies of that polynucleotide, unless defined herein and then in the remainder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by that of ordinary skill in the art with which the invention is concerned.

Orthogonal: As used herein, the term "orthogonal" refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and / or an orthogonal aminoacyl-tRNA N synthetase (O-RS)) that works with endogenous components of a cell with reduced efficiency compared to a corresponding molecule that is endogenous to the cell or translation system or that does not work with endogenous components of the cell. In the context of tRNA and aminoacyl-tRNA synthetases, orthogonal refers to a reduced disability or efficiency, for example, less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% of efficiency, from an orthogonal tRNA to function with an endogenous tRNA synthetase compared to an endogenous tRNA to function with the endogenous tRNA synthetase or from an orthogonal aminoacyl-tRNA synthetase to function with an endogenous tRNA compared to an endogenous tRNA synthase to function with the endogenous tRNA. The orthogonal molecule lacks a functionally normal endogenous complementary molecule in the cell. For example, an orthogonal tRNA in a cell is aminoacylated by an endogenous RS of the cell with reduced efficiency or even zero efficiency, when compared to the aminoacylation of an endogenous tRNA by the endogenous RS. In another example, an orthogonal RS aminoacylates any endogenous tRNA from a cell of interest with reduced efficiency or even zero efficiency, compared to the aminoacylation of endogenous tRNA by an endogenous RS. A second orthogonal molecule can be introduced into the cell that works with the first orthogonal molecule. For example, a pair of orthogonal tRNA / RS includes introduced complementary components that work together in the cell with an efficiency (eg, 45% efficiency, 50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency). efficiency, 80% efficiency, 90% efficiency, 95% efficiency, or 99% or more efficiency) compared to that of a control, for example, an endogenous pair of corresponding tRNA / RS or an active orthogonal pair (eg example, a pair of tirosil tARN / RS orthogonal).

Orthogonal tyrosyl-tRNA: As used herein, an orthogonal tyrosyl-tRNA (tyrosyl-O-tRNA) is a tRNA that is orthogonal to a translation system of interest, wherein the tRNA is: (1) identical or substantially similar to leucil or tyrosyl-tRNA that occurs stably in nature, (2) derived from a leucyl or tyrosyl-tRNA that is stably present in nature by natural or artificial mutagenesis, (3) derived by any process that takes into account a leucyl or tyrosyl-tRNA wild-type or mutant sequence of (1) or (2), (4), homologous to a leucyl or tyrosyl-tRNA wild-type or mutant; (5) homologous to any exemplary tRNA that is designed as a substrate for a leucyl or tyrosyl-tRNA synthetase in Table 5 or (6) a conservative variant of any exemplary tRNA that is designed as a substrate for a leucyl or tyrosyl-tRNA synthetase in Table 5. Leucyl or tyrosyl-tRNA may exist loaded with an amino acid or in an uncharged state. It will also be understood that a "tyrosyl-O-tRNA" or "leucyl-O-tRNA" is optionally charged (aminoacylated) by a cognate synthetase with an amino acid other than tyrosine or leucine, respectively, with an unnatural amino acid. Of course, it will be appreciated that a leucyl or tyrosyl-O-tRNA of the invention is advantageously used to insert essentially any amino acid, either natural or artificial, into a growing polypeptide, during translation, in response to a selector codon.

Tyrosyl amino acid synthetase orthogonal: As used herein, an orthogonal tyrosyl amino acid synthetase (tyrosyl -O-RS) is an enzyme that preferentially aminoacylates tyrosyl-O-tRNA with an amino acid in a translation system of interest. The amino acid that tyrosyl-O-RS charges to tyrosyl-O-tRNA can be any amino acid, whether natural, unnatural or artificial and is not limited in the present. The synthetase is optionally the same as or homologous with a tyrosyl amino acid synthetase which occurs stably in nature or the same as or homologous with a synthetase designed as O-RS in Table 4. For example, O-RS can be a conservative variant of a tyrosyl-O-RS of Table 4 and / or may be at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in sequence to an O-RS of Table 5. Similarly, an orthogonal leucyl amino acid synthetase (leucyl-O-RS) is an enzyme that preferably aminoacylates leucyl-O-tRNA with an amino acid in a translation system of interest. The amino acid that the leucyl-O-RS charges on the leucyl-O-tRNA can be any amino acid, whether natural, unnatural or artificial and is not limited in the present. The synthetase is optionally the same as or homologous with a leucyl amino acid synthetase which is stably present in nature or the same as or homologated with a synthetase designated as an O-RS in Table 5. For example, O-RS can be a conservative variant of a leucyl-O-RS of Table 5 and / or may be at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in sequence to an O-RS of Table 5.

Cognate: the term "cognate" refers to components that work together, for example an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase. The components can also be referred to as complementary.

Aminoacyla preferably: As used herein in reference to orthogonal translation systems, an O-RS "aminoacylates" a cognate O-tRNA when the O-RS charges O-tRNA with an amino acid more efficiently than any endogenous tRNA loads in an expression system. That is, when the O-tRNA and any given endogenous tRNA are present in a translation system in approximately equal molar proportions, the O-RS will load the O-tRNA more frequently than the endogenous tRNA will load. Preferably, the relative proportion of O-tRNA charged by the O-RS to the endogenous TARN loaded by the O-RS is high, preferably resulting in the O-RS loading the O-tRNA exclusively or almost exclusively, when the O- tRNA and endogenous tRNA are present in equal molar concentrations in the translation system. The relative proportion between O-tRNA and endogenous tRNA that is loaded by O-RS, when O-tRNA and O-RS are present at equal molar concentrations, is greater than 1: 1, preferably at least about 2: 1, more preferably 5: 1, still more preferably 10: 1, still more preferably 20: 1, still more preferably 50: 1, still more preferably 75: 1, still more preferably 95: 1, 98: 1, 99: 1, 100: 1, 500: 1, 1,000: 1, 5,000: 1 or greater. O-RS "preferably aminoacylates an O-tRNA with an unnatural amino acid" when (a) the O-RS preferably aminoacylates the O-tRNA as compared to an endogenous tRNA and (b) wherein said aminoacylations specific to the non-natural amino acid, as compared to the aminoacylation of O-tRNA by the O-RS with any natural amino acid. That is, when the unnatural and natural amino acids are present in equal molar amounts in a translation system comprising the O-RS and O-tRNA the O-RS will load the O-tRNA with the unnatural amino acid more frequently than with the natural amino acid. Preferably, the relative proportion of O-tRNA charged with the unnatural amino acid to O-tRNA charged with the natural amino acid is high. More preferably, O-RS charges the O-tRNA exclusively or almost exclusively, with the non-natural amino acid. The relative proportion between the charge of O-tRNA with the unnatural amino acid and the charge of O-tRNA with the natural amino acid, when both natural and non-natural amino acids are present in the system of deduction in equal molar concentrations, is greater than 1 : 1, preferably at least about 2: 1, more preferably 5: 1, still more preferably 10: 1, still more preferably 20: 1, still more preferably 50: 1, still more preferably 75: 1, still more preferably 95: 1, 98: 1, 99: 1, 100: 1, 500: 1, 1,000 : 1, 5,000: 1 or greater.

Codon selector: The term "selector codon" refers to codons recognized by O-tRNA in the translation process and not recognized by an endogenous tRNA. The anticodon loop of O-tRNA recognizes the selector codon on the mRNA and incorporates its amino acid, for example an unnatural amino acid, at this site in the polypeptide. Selector codes may include, for example, nonsense codons, such as retention codons, eg, amber, ocher and opal codons; codons of four or more bases; rare codons; codons derived from natural or unnatural base pairs and / or the like. suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, for example, by providing a mechanism for incorporating an amino acid into a polypeptide chain in response to a selector codon. For example, a suppressor tRNA can read through, for example, a retention codon (e.g., an amber, ocher or opal codon), a four-base codon, a rare codon, etc.

Suppression Activity: As used herein, the term "suppression activity" generally refers to the ability of tRNA (e.g., a suppressor tRNA) to allow translation reading of a codon (e.g., a codon) selector is an amber codon or a codon of 4 or more bases) otherwise it would result in translation completion or mistranslation (eg, frame shift). The suppression activity of tRNA suppression can be expressed as a percentage of observed translation reading activity compared to a second suppressor tRNA or compared to a control system, for example a control system lacking an O-RS. The present invention provides several methods by which the suppression activity can be quantified. The percent removal of a particular O-tRNA and O-RS against a selector codon (e.g., an amber codon) of interest refers to the percentage of expressed test marker activity given (e.g., LacZ), which includes a selector codon, in a nucleic acid encoding the expressed test marker, in a translation system of interest, wherein the translation system of interest includes an O-RS and an O-tRNA, as compared to a control construct positive, where the positive control lacks the O-tRNA, the O-RS and the selector codon. So, for example, if an active positive control marker construct lacks a selector codon has an observed activity of X in a given translation system, in units relevant to the analysis of the marker in question, then the percent of deletion of a test construct comprising the selector codon is the percentage of X that the marker construct of test shows under essentially the same environmental conditions as under which the positive control marker was expressed, except that the test marker construct is expressed in a translation system that also includes the O-tRNA and the O-RS. The translation system that expresses the test marker also includes an amino acid that is recognized by the O-RS and the O-tRNA. Optionally, the measurement of percent suppression can be refined by comparing the test marker with a marker construct. "background" or "negative" control, which includes the same selector codon as the test marker, but in a system that does not include the 0-tRNA, O-RS and / or relevant amino acid recognized by the O-tRNA and / or the O-RS. This negative control is useful for normalizing the percent suppression measurements to take into account the background signal effect of the marker in the translation system of interest. The suppression efficiency can be determined by any number of analyzes known in the art. For example, a reporter analysis of β-galactosidase can be used, for example a derivative lacZ plasmid (wherein the construct has a selector codon n in the lacZ nucleic acid sequence) is introduced into cells of an appropriate organism (eg, an organism in which the orthogonal components can be used) together with the plasmid comprising an O-tRNA of the invention . A cognate synthetase can also be introduced (either as a polypeptide or a polynucleotide encoding the cognate synthetase when it is expressed). The cells are cultured in media at a desired density for example, at OD60o of about 0.5, and analyzes of β-galactosidase are carried out, for example using the BetaFluor ™ beta-galactosidase analysis kit.

(Novagen). The percent of suppression can be calculated as the percentage of activity for a sample relative to a comparable control, for example the observed value of the derived lacZ construct, wherein the construct has a corresponding sense codon at a desired position instead of a selector codon.

Translation system: the term "translation system" refers to components that incorporate an amino acid into a growing polypeptide chain (proteins). The components of a translation system may include, for example, ribosomes, tRNAs, synthetases, mRNA and the like. The O-tRNA and / or the O-RSs of the invention can be added or be part of an in vitro or in vivo translation system, by example, in a non-eukaryotic cell, for example a bacterium (such as E. coli) or in a eukaryotic cell, for example, a yeast cell, a mammalian cell, a plant cell, an algae cell, a cell fungi, an insect cell and / or the like.

Non-natural amino acid - As used herein, the term "non-natural amino acid" refers to any amino acid, modified amino acid and / or amino acid analogue, such as an alkyl amino acid, which is not one of the 20 amino acids that are presented Stably in the common nature or selenocysteine or pirrolisma. For example, Figure 1 provides 17 non-natural amino acids that find use with the invention.

Derived from: As used herein, the term "derived from" refers to a component that is isolated from or manufactured using a specified molecule or organism or information from the specified molecule or organism. For example, a polypeptide that is derived from a second polypeptide comprises an amino acid sequence that is identical or substantially similar to the amino acid sequence of the second polypeptide. In the case of polypeptides, the derived species can be obtained, for example, by mutagenesis that is presented in a stable manner. in nature, artificially directed mutagenesis or artificial random mutagenesis. The mutagenesis used to derive polypeptides can be purposely directed or intentionally randomized. The mutagenesis of a polypeptide to create a different polypeptide derived from the former may be a random event (eg, caused by polymerase infidelity) and the identification of the derived polypeptide may be successful. Mutagenesis of a polypeptide commonly encompasses the manipulation of the polynucleotide encoding the polypeptide.

Selection Marker or Positive Filtration: As used herein, the term "selection marker or positive filtration" refers to a label which, when present, for example expressed, activated or the like, results in the identification of a cell, which comprises the trait, for example cells with the positive selection marker, of those without the trait.

Negative selection marker or negative selection marker: As illustrated herein, the term "negative selection marker or selection marker" refers to a marker which, when present, eg expressed, activated or the like, allows the identification of a cell that does not understand a property or trait selected (for example, compared to a cell that owns the property or trait).

Reporter: As used herein, the term "reporter" refers to a component that can be used to identify and / or select target components of a system of interest. For example, a reporter may include a protein, for example an enzyme, which confers antibiotic resistance or sensitivity (eg, β-lactamase, chloramphenicol acetyltransferase (CAT), and the like), a fluorescent selection marker (eg, fluorescent protein) green (e.g., (GFP), YFP, EGFP, RFP, etc.), a luminescent marker (e.g., a firefly luciferase protein), an affinity-based selection marker, or selectable positive or negative marker genes such as lacZ, ß-gal / lacZ (ß-galactosidase), ADH (alcohol dehydrogenase), his3, ura3, leu2, lys2, or the like.

Eukaryota: As used in the present, the term "eukaryote" refers to organisms belonging to the kingdom of Eucarya. Eukaryotes are generally distinguishable from prokaryotes by their commonly multicellular organization (but not exclusively multicellular, for example, yeast), the presence of a membrane-bound core and other organelles membrane-bound, linear genetic material (ie, linear chromosomes), the absence of operons, the presence of introns, messenger coronation and poly -A mRNA and other biochemical features, such as a distinctive ribosomal structure. Eucaponeic organisms include, for example, animals (eg, mammals, insects, reptiles, birds, etc.), ciliates, plants (eg, monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microspopdia, protists, etc Prokaryote • As used herein, the term "prokaryote" refers to organisms belonging to the kingdom of Monera (also called Procarya). Prokaryotic organisms are generally distinguishable from eukaryotes because of their unicellular organization, asexual reproduction by budding or fission, the lack of a nucleus linked to the membrane or other organelles linked to the membrane, a circular chromosome, the presence of operons, the absence of mtrons, coronation of message and poly-A mRNA and other biological characteristics, such as a distinctive ribosomal structure. The prokaryotes include Subacteria submalaria and Archaea (sometimes called "Archaebacteria"). The cyanobacteria (blue green algae) and mycoplasma are sometimes given separate classifications under the kingdom of Monera.

Bacteria: As used herein, the terms "bacteria" and "eubacteria" refer to prokaryotic organisms that are distinguishable from Archaea. Similarly, Archaea refers to prokaryotes that are distinguishable from eubacteria. Eubacteria and Archaea can be distinguished by a variety of morphological and biochemical criteria. For example, differences in ribosomal RNA sequences, RNA polymerase structure, the presence or absence of introns, antibiotic sensitivity, the presence or absence of cell wall peptidoglycans and other cell wall components, the branched structures against unbranched membrane lipids and / or the presence / absence of histones and histone-like proteins are used to assign an organism to an Eubacteria or Archaea. Examples of Eubacteria include Escherichia coli, Thermus thermophilics and Bacillus stearother ophilus. Examples of Archaea include Methanococcus jannaschii (Mj), Methanosarcina azei (Mm), Methanobacteriu thermoautotrophicum (Mt), Methanococcus maripaludis, Methanopyrus kandleri, Halobacterium such as Haloferax volcanii and Halobacterium NRC-I, Archaeoglobus fulgidus (Af), Pyrococcus furiosus (Pf), Pyrococcus horikoshii (Ph), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ss) , Sulfolobus tokodaii, Aeuropyrum pernix (Ap), Ther oplasma acidophilum and Thermoplasma volcanium.

Conservative variant: As used in this, the term "conservative variant," in the context of a translation component, refers to a translation component, for example, a conservative O-tRNA variant or a conservative O-RS variant, which behaves functionally similar to a component base that the conservative variant is similar, for example an O-tRNA or O-RS, which has variations in the sequence with a reference O-tRNA or O-RS. For example, an O-RS or a conservative variant of that O-RS will aminoacilate a cognate O-tRNA with an unnatural amino acid, for example an amino acid comprising a portion of N-acetylgalactosamine. In this example, the O-RS and the O-RS of conservative variant do not have the same amino acid sequences. The conservative variant may have, for example, one variation, two variations, three variations, four variations or five or more variations in sequence, as long as the conservative variant is still complementary with the corresponding O-tRNA or O-RS. In some embodiments, an O-RS of conservative variant comprises one or more conservative amino acid substitutions as compared to the O-RS from which it was derived. In some embodiments, an O-RS of conservative variant comprises one or more conservative amino acid substitutions compared to the O-RS from which it was derived, and in addition, retains biological activity of O-RS.; by example, an O-RS of a conservative variant that retains at least 10% of the biological activity of the original O-RS molecule from which it was derived, or alternatively, at least 20%, at least 30% or at least 40% In some preferred eiments, the preservative variant O-RS retains at least 50% of the biological activity of the original O-RS molecule from which it was derived. Conservative amino acid substitutions of a conservative variant O-RS can occur in any domain of the O-RS, in which the amino acid binding cavity is included.

Selection or filtering agent: As used herein, the term "selection or filtering agent" refers to an agent that, when present, allows the selection / filtration of certain components of a population. For example, a screening or filtering agent can be, but is not limited to, for example a nutrient, an antibiotic, a wavelength of light, an antibody, an expressed polynucleotide or the like. The selection agent can be varied, for example by concentration, intensity, etc.

In response to: As used herein, the term "in response to" refers to a process in which an O-tRNA of the invention recognizes a selector codon and moderates the incorporation of the alkynyl amino acid, which is coupled to the tRNA, to the growing polypeptide chain.

Encode: As used herein, the term "encodes" refers to any process by which information in a polymer macromolecule or sequence chain is used to direct the production of a second molecule or sequence chain that is different from the first molecule or sequence chain. As used herein, the term is widely used and may have a variety of applications. In one aspect, the term "coding" describes the process of replication of semiconservative DNA, wherein a strand of a double-stranded DNA molecule is used as a template to encode a complementary sister strand newly synthesized by a DNA-dependent DNA polymerase. In another aspect, the term "encodes" refers to any process by which information in a molecule is used to direct the production of a second molecule that has a different natural chemistry from the first molecule. For example, a DNA molecule can encode an RNA molecule (for example, by the transcription process that incorporates a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the translation process. When used to describe the translation process, the term "coding" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, for example by the process of reverse transcription that incorporates an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, wherein it is understood that "coding" as used in that case incorporates both the transcription and translation processes.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A provides the chemical structures of several non-natural amino acids. Figure 2 provides a photograph of a stained SDS-PAGE analysis of the accumulated Z domain protein in the presence (lane 2) or absence (lane 3) of p-nitro-L-phenylalanine. Lane 1 contains molecular mass markers. Figure 3 provides a MALDI-TOF analysis of Z domain protein incorporated into p-nitro-L-phenylalanine. Expected mass: 7958, 7826 (exclusion of first methionine); Observed: 7958, 7828. Figure 4A provides fluorescence spectra of the 22Trp GCN4pl mutant (solid lines) and the mixture of 22Trp and 22p-nitro-L-phenylalanine GCN4pl mutants (dotted lines). Figure 4B provides spectra of fluorescence of the 55Trp GCN4pl mutant (solid line) and the mixture of 55Trp and GCN4pl 22p-nitro-L-phenylalanine mutants (dotted lines). Figure 5A provides the chemical structure of 1, 5-dansilalanine. Figure 5B provides an active site model of leucyl-tRNA synthetase (LRS) from Thermus ther ophilus with 1,5-dansilalanine-bound AMP-amide. The active site residues of the mutant LRS clone B8 that are part of the randomized region are shown as bars. The numbering corresponds to LRS of E. coli. Figures 6A and 6B describe the redesign strategy of the mutant leucyl-tRNA synthetase B8 editing site. Figure 6A provides the crystal structure of the leucyl-tRNA synthetase editing site of Ther us thermophilus in complex with 2 '- (1-norvalyl) -amino-2'-deoxyadenosine which mimics the 3' term of charged tRNA . T252 and V340 are shown as bars. Figure 6B provides analysis of SDS-PAGE analysis of dansilalanin carrying Ni-NTA purified with hSOD at position 33 using the con of leucyl-tRNA synthetase B8 and the two mutants V338A and T252A. The upper gel is a photograph of a Coomassie stain. The lower gel a fluorescence image with excitation at 302 nm and emission detection at 520 nm. L = molecular weight ladder; UAA = non-natural amino acid. Figures 7A and 7B describe the efficiency of improved amber suppression of leucyl-tRNA synthetase clone of E. coli G2-6, generated by PCR prone to error and selection. Figure 7A provides the crystal structure of the leucyl-tRNA synthetase Thermus thermophilus (Cusack et al., EMBO J., 19 (10): 2351-2361 [2000]). The synthetic domain, editing domain, randomized amino acids in the homologous E. coli synthetase and PCR-prone amino acid exchanger in the clone G2-6 are all indicated. Figure 7B provides an SDS-PAGE analysis of Coomassie staining of o-nitrobenzyl serine carrying hSOD expressed at position 33 using the mutant leucyl-tRNA synthetase clone of E. coli 3H11 designated for incorporation of o-nitrobenzylcysteine and the clone of leucyl-tRNA synthetase from mutant E. coli G2-6 evolved for efficient suppression with o-nitrobenzyl serine. L = molecular weight ladder; UAA = non-natural amino acid (oNBS). Figure 8 provides a schematic representation of the photodesynated (photoactivation) of the caged tyrosine molecule O- (2-nitrobenzyl) -L-tyrosine by irradiation at 365 nm, resulting in cleavage of the benzylic CO bond and rapid formation of the amino acid unattached. Figure 9 provides concentration curve analysis illustrating the experimentally observed photodesynjunction (photoactivation) of the tyrosine molecule caged O- (2-nitrobenzyl) -L-tyrosine. The O-(2-nitrobenzyl) -L-tyrosine photodensation was illustrated by irradiation of a 0.2 mM amino acid solution in water using a portable ultraviolet lamp (365 nm at 10 mm distance). Aliquots were formed at specific time points and analyzed by LC / MS. The concentrations of 0- (2-nitrobenzyl) -L-tyrosine (square) and the corresponding de-entrained species (circles) are shown. Figure 10 provides a SDS-PAGE stained with Gelcode blue of 74TAG myoglobin expressed in the presence or absence of O- (2-nitrobenzyl) -L-tyrosine using three different mutant synthetases. Figures HA and 11B provide an LC-MS / MS analysis of 74TAG mutant myoglobin protein showing tyrosine at position 74 (tryptic peptide HGVTVLTALGYILK). Figures 12A and 12B provide an LC-MS / MS analysis similar to that described in Figures HA and 11B, except where the analysis uses a deuterated O- (2-nitrobenzyl) -L-tyrosine, where J denotes the deuterated amino acid (tryptic peptide HGVTVLTALGJILK). Figure 13 provides graphs of superposition of IR spectra of para-cyanophenylalanine taken in THF and water. Figure 14A provides the subtracted spectrum of the para-cyanophenylalanine background (the solid) adjusted to a Gaussian curve (dashed lines). Figure 14B provides the spectrum subtracted from the meta-cyanophenylalanine background (solid lines) adjusted to two Gaussian curves (discontinuous). Figure 15 provides a scheme describing the synthesis of p-ethylthiocarbonyl-L-phenylalanine. Figure 16 provides a result of mass spectrum analysis of MALDI-TOF of mutant Z-domain proteins containing unnatural amino acid in the seventh position. All experimentally obtained mass data are in excellent agreement with those calculated masses of intact proteins that have either thioester or carboxylic acid groups. Figure 17 provides a protein scheme by chemical ligation. Figures 18A-18D provide LC / MS elusion profiles (first peak: 3; second peak: 2) verified at 340 nm. Figure 18A shows a profile using a 1: 1 mixture of 3 and 2 in MeOH. Figure 18B shows a profile using 3 in PBS (pH = 7.4, reaction time: 1 week). Figure 18C shows a profile using 3 in PBS (pH = 3.9, reaction time: 4 days). Figure 18D shows a profile using 3 in dilute H2SO4 solution (pH = 1.9, reaction time: 12 hours). All reactions were carried out at room temperature with constant stirring. Figure 19 provides a schematic describing the synthesis of the non-natural amino acid p- (3-oxobutanoyl) -L-phenylalanine containing diketone. Figure 20 provides an analysis of SDS-PAGE stained with Gelcode blue of Z domain protein expressed in the presence or absence of p- (3-oxobutanoyl) -L-phenylalanine. The analysis shows the in vitro labeling of the mutant Z domain protein containing p- (3-oxobutanoyl) -L-phenylalanine with fluorescein hydrazide. wt = wild type. Figure 21 provides a scheme describing the synthesis of various adducts of the diketone-containing portion.

DETAILED DESCRIPTION OF THE INVENTION The invention provides solutions to the inherent limitations of using a translation system confined by the twenty amino acids that occur stably in nature. The solutions include the biosynthetic incorporation specific to the programmed site of non-natural amino acids with new properties to proteins using orthogonal translation systems. New compositions are described herein (e.g., new amino acid-tRNA synthetases) and new methods for the highly efficient and specific genetic incorporation of a variety of non-natural amino acids into proteins in response to a selector codon (e.g. amber sense, TAG).

In some cases, the unnatural amino acid side chains can then be modified specifically and regioselectively. Due to the unique reaction chemistries of these non-natural amino acid substituents, the proteins to which they are incorporated can be modified with extremely high selectivity. In some cases, the unnatural amino acid reactive group has the advantage of being completely foreign to the in vivo systems, thereby improving the selectivity of the reaction. In some aspects, the modification reactions can be carried out using relatively moderate reaction conditions that allow both in vitro and in vivo conjugation reactions involving proteins and preserving the biological activity of protein. The nature of the material is conjugated to an unnatural amino acid in a protein is not particularly limited and can be any desired entity, for example dyes, fluorophores, crosslinking agents, saccharide derivatives, polymers (for example polyethylene glycol derivatives), photo-crosslinking, cytotoxic compounds, affinity tags, biotin derivatives, resins, beads, a second protein or polypeptide (or more), polynucleotide (s) (eg DNA, RNA, etc.), metal chelators, co-factors , fatty acids, carbohydrates and the like. In other aspects, the non-natural amino acid incorporated impart new biological properties to the protein to which it is incorporated. For example, the non-natural amino acid can be a fluorescent amino acid, a photoenaged or photoactivatable amino acid, an amino acid that can participate in a FRET pair as a donor or acceptor, a redox-active amino acid, a metal chelating amino acid, etc. In some aspects, to demonstrate (but not limit) the present invention, the disclosure herein demonstrates that the non-natural amino acid portion can be incorporated into a model protein. The incorporation of the non-natural amino acid is not intended to be limited to such model protein. From the present disclosure, it will be clear that the incorporation of an unnatural amino acid into any given protein of interest is advantageous for a wide variety of proteins for use in therapeutic and research purposes. New orthogonal tRNA / aminoacyl-tRNA synthetase pairs have been developed that function in eubacteria and yeast to specifically incorporate non-natural amino acids (eg, the unnatural amino acids provided in Figure 1) into the site in response to codons selectors. Briefly, new mutants of the tyrosyl-tRNA synthetase of Methanococcus jannaschii and the leucyl-tRNA synthetase of Escherichia coli have been identified that selectively load a suppressor tRNA with an unnatural amino acid either in E. coli host cells or host cells of yeast, respectively. These developed tRNA synthetase pairs can be used to specifically incorporate at the site the respective non-natural amino acid to a protein. Incorporation of the non-natural amino acid into the protein can be programmed to occur at any desired position by designing the polynucleotide encoding the protein of interest to contain a selector codon signaling the incorporation of the non-natural amino acid.

TUNN / AMINOACILTARN TECHNOLOGY ORTHOGONAL SYNTHETASE An understanding of the new compositions and methods of the present invention is facilitated by an understanding of the activities associated with orthogonal tRNAs and ammoacyl-tRNA orthogonal smtetase. Discussions of orthogonal and ammoacyte tRNA technologies tARN smtetase can be found, for example, in international publications WO 2002/085923, WO 2002/086075, WO 204/09459, WO 2005/019415, WO 2005/007870 and WO 2005/007624. See also, Wang and Schultz "Expandmg the Genetic Code", Angewandte Chemie Int. Ed., 44 (l): 34-66 (2005), the content of which is incorporated herein by reference in its entirety. In order to add additional non-natural amino acids reactive to the genetic code, new orthogonal pairs comprising an aminoacyl-tRNA synthetase and an appropriate tRNA that can function efficiently in the host translation machinery, but that are "orthogonal" to the translation system in question, which means that they work independently of the endogenous synthetases and tRNAs in the translation system. The desired features of the orthogonal pair include tRNA that decodes or recognizes only a specific codon, for example a selector codon, which is not decoded by any endogenous tRNA, an ammoacyl-tRNA smtetase that preferentially (or "charges") its cognate tRNA with only a specific non-natural amino acid. O-tRNA is also not commonly ammoacylated by endogenous smtetases. For example, in E. coli, an orthogonal pair will include an aminoacylTARN smtetase that does not cross-react with any of the endogenous tRNAs, for example, which are 40 in E. coll and an orthogonal tRNA that is not ammoacylated by either of endogenous smtetases, for example, of which they are 21 in E. coli. The invention described herein provides orthogonal pairs for the genetic coding and incorporation of non-natural amino acids into proteins in a eubacteria, for example an E. coli or in yeast, where the orthogonal components do not cross-react with the E components. Endogenous coli or yeast from the translation machinery of the host cell, but recognize the amino acid desired non-natural and incorporate it into proteins in response to the selector codon (eg, a non-sense amber codon, TAG). The orthogonal components provided by the invention include orthogonal aminoacyl-tRNA synthetases derived from tyrosyl tRNA synthetase from Methanococcus jannaschii and the amber suppressor from mutant tyrosyl tARNCUA, which function as an orthogonal pair in a eubacterial host cell. The invention also provides orthogonal components derived from leucyl-tRNA-synthetase from E. coli and an amber suppressant from leucyl tARNCUA from mutant E. coli, which function as an orthogonal pair in a yeast host cell. In these systems, the mutant aminoacyl-tRNA synthetases aminoacylate the suppressor tRNA with its respective non-natural amino acid and not with any of the twenty common amino acids. The invention provides compositions and methods for identifying and producing additional orthogonal tRNA-aminoacyl-tRNA synthetase pairs, for example, 0-tRNA / O-RS pairs that can be used to incorporate an unnatural amino acid into a protein. A pair of O-ARNA / O-RS of the invention is capable of moderating the incorporation of a non-natural amino acid, eg, a non-natural amino acid shown in Figure 1, into a protein that is encoded by a polynucleotide, wherein the polynucleotide comprises a selector codon which is recognized by the O-tRNA, for example, in vivo. The anticodon loop of the O-tRNA recognizes the selector codon on a mRNA and incorporates its amino acid, for example a non-natural amino acid shown in Figure 1, at this site in the polypeptide. In general, an orthogonal aminoacyl-tRNA synthetase of the invention preferably aminoacylates (or charges) its O-tRNA with only a specific non-natural amino acid. The ability to incorporate a non-natural amino acid (eg, a non-natural amino acid provided in Figure 1) specifically at the protein site can facilitate the study of proteins, as well as enable the design of proteins with new properties. For example, the expression of proteins containing one or more non-natural amino acids may facilitate the study of proteins by specific labeling, alter the catalytic function of enzymes, improve biological activity or reduce cross-activity to a substrate, cross-linking a protein with other proteins, small molecules or biomolecules, reducing or eliminating protein degradation, improving the half-life of proteins in vitro (for example, by means of pegylation or other modifications of introduced reactive sites), etc.

ORTOGONAL TART / AMINOACIL-TARN ORTHOGONAL SYNTHETICS AND PAIRS OF THEMSELVES Translation systems that are suitable for making proteins that include one or more non-natural amino acids are described, for example, in the international publication WO 2002/086075, entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINO ACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" and WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415, filed July 7, 2004; WO 2005/007870, filed July 7, 2004 and WO 2005/007624, filed July 7, 2004. Each of these applications is incorporated herein by reference in its entirety. See also Wang and Schultz "Expanding the Genetic Code", Angewandte Chemie Int. Ed. , 44 (1): 34-66 (2005), the content of which is incorporated by reference in its entirety. Such translation systems generally comprise cells (which may be non-eukaryotic cells such as E. coli or eukaryotic cells such as yeast) that include an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl tRNA N synthetase (0-RS), and a non-natural amino acid, where O-RS aminoacylates O-tRNA with the non-natural amino acid. An orthogonal pair of the invention includes an O-tRNA, for example, a suppressor tRNA, a frame shift tARN or the like and an O-RS. Individual components are also provided in the invention. In general, when an orthogonal pair recognizes a selector codon and loads an amino acid in response to the selector codon, it is said that the orthogonal pair "suppresses" the selector codon. This is, a selector codon that is not recognized by the endogenous machine of the translation system (eg, of the cell) is not translated ordinarily, which may result in production blockage of a polypeptide that would otherwise be translated from the acid nucleic. An O-tRNA of the invention recognizes a selector codon and includes at least about, for example, 45%, 50%, 60%, 75%, 80%, or 90% or more suppression efficiency in the presence of a cognate synthetase in response to a selector codon compared to the efficiency of deletion of a 0-tRNA that comprises or is encoded by a polynucleotide sequence as summarized in the sequence listing herein. O-RS aminoacylates O-tRNA with an unnatural amino acid of interest. The cell uses the 0-tRNA / 0-RS pair to incorporate the unnatural amino acid into a growing polypeptide chain, for example via a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a codon. selector that is recognized by the O-tRNA. In certain desirable aspects, the cell may include a pair of additional O-tRNA / 0-RS, wherein the additional O-tRNA is loaded by the additional O-RS with a different unnatural amino acid. For example, one of the 0-tRNA may be a four-base codon and the other may recognize a retention codon. Alternatively, multiple codons of different retention or multiple codons of four Different bases can specifically recognize different codons selectors. In certain embodiments of the invention, a cell such as an E. coli cell or a yeast cell that includes an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl-tRNA N synthetase (O-RS), an unnatural amino acid and a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the O-tRNA. The translation system can also be a cell-free system, for example, any of a variety of commercially available "in vitro" transcription / translation systems in combination with a pair of O-tRNA / O-RS and an unnatural amino acid as described herein. In one embodiment, the suppression efficiency of the 0-RS and the O-tRNA together is approximately, for example, 5 times, 10 times, 15 times, 20 times, or 25 times or greater than the efficiency of O suppression. -tRNA that lacks the O-RS. In one aspect, the suppression efficiency of the O-RS and the O-tRNA together is at least approximately, for example, 35%, 40%, 45%, 50%, 60%, 75%, 80%, or 90% or more of the efficiency of suppression of an orthogonal synthetase pair as summarized in the sequence listings herein. As indicated, the invention optionally includes multiple pairs of O-tRNA / O-RS in a cell or other system of translation, which allows the incorporation of more than one non-natural amino acid. For example, the cell may further include a further different pair of O-tRNA / O-RS and a second unnatural amino acid, wherein this additional O-tRNA recognizes a second selector codon and this additional O-RS preferably aminoacylates the O- tRNA with the second non-natural amino acid. For example, a cell including a pair of O-tRNA / O-RS (wherein the O-tRNA recognizes, for example, an amber selector codon), may further comprise a second orthogonal pair, wherein the second O-tRNA recognizes a different selector codon, for example, an opal codon, a four-base codon or the like. Desirably, the different orthogonal pairs are derived from different sources, which may facilitate the recognition of different codons selectors. The O-tRNA and / or the O-RS can occur in a stable manner in nature, or they can, for example, be derived by mutation of a tRNA and / or RS that is stably present in nature, for example when generating tRNA libraries and / or RS libraries, from any of a variety of organisms and / or by using any of a variety of available mutation strategies. For example, a strategy for producing a pair of orthogonal tRNA / aminoacyl-tRNA synthetase involves importing a pair of heterologous tRNA / synthetase (to the host cell) of for example, a source different from the host cell or multiple sources, to the cell Guest. The properties of the candidate heterologous synthetase include, for example, that it does not load any host cell tRNA and the properties of the candidate heterologous tRNA include, for example, that it is not aminoacylated by any host cell synthetase. In addition, the heterologous tRNA is orthogonal to all host cell synthetases. A second strategy for generating an orthogonal pair involves generating mutant libraries from which to filter and / or select an O-tRNA or O-RS. These strategies can also be combined. orthogonal tRNA (O-tRNA) an orthogonal tRNA (O-tRNA) of the invention desirably moderates the incorporation of an unnatural amino acid into a protein that is encoded by a polynucleotide comprising a selector codon that is recognized by O-tRNA, for example in vivo or in vitro. In certain embodiments, an O-tRNA of the invention includes at least, for example, about 45%, 45%, 50%, 60%, 75%, 80%, or 90% or more suppression efficiency in the presence of a cognate synthetase in response to a selector codon compared to an O-tRNA that comprises or is encoded by a polynucleotide sequence as summarized in the O-tRNA sequences in the sequence state herein.

The suppression efficiency can be determined by any of a number of assays known in the art. For example, a reporter analysis of ß-galactosidase can be used, for example a derived lacZ plasmid (wherein the construct has a n-codon in the nucleic acid sequence) is introduced into cells of an appropriate organism (e.g. an organism in which the orthogonal components can be used) together with a plasmid comprising an O-tRNA of the invention. A cognate synthetase can also be introduced (either as a polypeptide or a polynucleotide encoding the cognate synthetase when expressed). The cells are cultured in media at a desired density for example, at OD60o of about 0.5, and analyzes of β-galactosidase are performed, for example, using the beta-galactosidase analysis BetaFluor ™ (Novagen). The percent of suppression can be calculated as the percentage of activity for a sample relative to a comparable control, for example, the observed value of the derived lacZ construct, wherein the construct has a corresponding sense codon in the desired position instead of a selector codon. Examples of O-tRNA of the invention are summarized in the sequence listing herein. See also tables, examples and figures herein for 0-tRNA sequences and exemplary O-RS molecules. See also, the section entitled "Nucleic acid and polypeptide sequence and variants" herein. In an RNA molecule, such as an O-RS mRNA, or an O-tRNA molecule, thymine (T) is replaced with uracil (U) in relation to a given sequence (or vice versa for a coding DNA) or a complement of it. Additional modifications to the bases may also be present. The invention also includes conservative variations of O-tRNA corresponding to particular O-tRNAs herein. For example, conservative variants of O-tRNA include those molecules that function as the particular O-tRNA, for example as in the listing of corresponding sections and which maintain the L-shaped structure of tRNA under the appropriate self-complementation; but which do not have an identical sequence to those, for example, in the sequence listing, figures or examples herein (and desirably, are different from the wild-type tRNA molecules). See also, the section in the present entitled "Nucleic acids and sequence of polypeptides and variants". The composition comprising an O-tRNA may further include an orthogonal aminoacyl-tRNA N synthetase (O-RS), wherein the O-RS preferably aminoacylates the O-tRNA with an unnatural amino acid. In certain embodiments, a composition that includes an O-tRNA may also include a translation system (e.g., in vitro or in vivo). A nucleic acid comprising a polynucleotide that encodes a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the O-tRNA or a combination of one or more of these may also be present in the cell. See also, the section in the present entitled "aminoacyl-tRNA orthogonal synthetases". Methods for producing an orthogonal tRNA (O-tRNA) also an aspect of the invention. An O-tRNA produced by the method is also an aspect of the invention. In certain embodiments of the invention, the O-tRNAs can be produced by generating a library of mutants. The mutant tRNA library can be generated using various mutagenesis techniques known in the art. For example, mutant tRNAs can be generated by site-specific mutations, random point mutations, homologous recombination, DNA redistribution or other methods of recursive mutagenesis, chimeric construction or any combination thereof, for example, of the exemplary O-tRNA. from table 5. Additional mutations can be introduced in a specific position (s), for example in a non-conservative position (s) or in a conservative position in one (s) randomized position (s) or a combination of both in a desired loop or region of a tRNA, eg, an anticodon loop, the acceptor stem, arm or loop D, variable loop, arm or TPC loop, other regions of the molecule of tRNA or a combination thereof. Commonly, mutations in a tRNA include mutation of the anticodon loop of each library member of the mutant tRNA to allow recognition of a selector codon. The method may also include adding additional sequences to the O-tRNA. Commonly, an O-tRNA has an orthogonality enhancement for a desired organism compared to the starting material, for example the plurality of tRNA sequences, while retaining its affinity for a desired SR. The methods optionally include analyzing the similarity (and / or inferred homology) of tRNA and / or aminoacyl-tRNA synthetases sequences to determine potential candidates for an O-tRNA, O-RS and / or pairs thereof, which appear to be orthogonal for a specific organism. Computer programs known in the art and described herein may be used for analysis, for example BLAST and stacking programs may be used. In one example, to choose potential orthogonal translation components for use in E. coli, a synthetase and / or a tRNA is chosen that does not show close sequence similarity to eubacterial organisms. Commonly, an O-tRNA is obtained by subjecting, for example, to negative selection, a population of cells of a single species, wherein the cells comprise a member of the plurality of potential O-tRNAs. The negative selection eliminates cells comprising a member of the potential O-tRNA library that is aminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous to the cell. This provides a cluster of tRNAs that are orthogonal to the cell of the first species. In certain embodiments, in the negative selection, a selector codon (s) is (are) introduced to a polynucleotide that encodes a negative solution marker, for example an enzyme that confers antibiotic resistance, for example β-lactamase, an enzyme that confers a detectable product, for example β-galactosidase, chloramphenicol acetyltransferase (CAT), for example, a toxic product, such as barnase, in a non-essential position (eg, still producing a functional barnase), etc. Filtration / selection is optionally done by culturing the cell population in the presence of a selective agent (e.g., an antibiotic, such as ampicillin). In one embodiment, the concentration of the selection is varied. For example, to measure the activity of suppressor tRNAs, a selection system is used which is based on the in vivo deletion of the selector codon, for example nonsense mutations (eg retention) or frame shift mutations introduced into a polynucleotide that encodes a negative selection marker, for example a gene for β-lactamase (bla). For example, variants of polynucleotide, for example bla variants, with a selector codon at a certain position (for example A184) are constructed. Cells, for example bacteria, are transformed with these polynucleotides. In the case of an orthogonal tRNA, which can not be efficiently loaded by endogenous E. coli synthetases, resistance to the antibiotic, for example resistance to ampicillin, should be approximately or less than that for a bacterium transformed without plasmid. If the tRNA is not orthogonal or if a heterologous synthetase capable of loading the tRNA is co-expressed in the system, a higher level of antibiotic resistance will be observed, for example ampicillin. Cells, for example bacteria, are chosen that are not suitable for growth on LB agar boxes with antibiotic concentrations and approximately equal to transformed cells without plasmid. In the case of a toxic product (e.g., ribonuclease or barnase), when a member of the plurality of potential tRNAs is aminoacylated by the endogenous host, for example, Escheri chia coli synthetases (ie, it is not orthogonal to the host, example, Escherichia coli synthetases), the selector codon is deleted and the toxic polynucleotide product produced leads to cell death. Cells harboring orthogonal tRNAs or non-functional tRNAs survive. In one modality, the cluster of tRNAs that are orthogonal to a desired organism are then subjected to a positive selection in which a selector codon is placed in a positive selection marker, for example encoded by a drug resistant gene, such as a ß-lactamase gene. The positive selection is effected on a cell comprising a polynucleotide that encodes or comprises a member of the tRNA cluster that is orthogonal to the cell, a polynucleotide encoding a positive selection marker, and a polynucleotide encoding a cognate RS. In certain embodiments, the second cell population comprises cells that were not eliminated by the negative selection. The polynucleotides are expressed in the cell and the cell is cultured in the presence of a selection people, for example ampicillin. Then tRNA is selected for its ability to aminoacylate co-expressed cognata synthetase and to insert an amino acid in response to this selector codon. Commonly, these cells show an improvement in suppression efficiency compared to cells harboring non-functional tRNAs or tRNAs that can not be efficiently recognized by the synthetase of interest. The cell harboring the non-functional tRNA or tRNA that are not efficiently recognized by the synthetase of interest are sensitive to the antibiotic. Accordingly, tRNA that: (i) are not substrates for the endogenous host, for example Escherichia coli, synthetases; (ii) can be aminoacylated by the synthetase of interest and (iii) are functional in translation, about live to both selections. Thus, the same marker can be either a positive marker or a negative marker, depending on the context in which it is selected. That is, the marker is a positive marker if it is selected for, but a negative marker if it is selected against. The severity of the selection, for example positive selection, negative selection or both positive and negative selection, in the methods described above, optionally includes varying the severity of selection, for example, because barnase is an extremely toxic protein , the severity of the negative selection can be controlled by introducing different numbers of selector codons in the barnase gene and / or by using an inducible promoter. In another example, the concentration of the selection or filtration people is varied (e.g., ampicillin concentration). In one aspect of the invention, the severity is varied because the desired activity may be low during the early rounds. Thus, less severe selection criteria are applied in the early rounds and more severe criteria are applied in later rounds of selection. In certain modalities, negative selection, positive selection or both negative selection and positive selection can be repeated multiple times. Multiple different negative selection markers, positive selection markers or both negative and positive selection markers or they can be used. In certain modalities, the positive and negative selection marker may be the same. Other types of selections / filtrations can be used in the invention to produce orthogonal translation components, for example an O-tRNA, an O-RS and a pair of 0-tRNA / 0-RS that charges an unnatural amino acid in response to a selector codon For example, the negative selection marker, the positive selection marker or both positive, negative selection markers may include a marker that fluoresces or catalyzes a luminescent reaction in the presence of an appropriate reagent In another embodiment, a marker product is detected by fluorescence activated cell sorting (FACS) or by luminescence Optionally, the marker includes an affinity-based selection marker. See also Francisco, JA, et al, (1993) Production and fl uorescence-active ted cell sorting of Eschep chia coll expressmg a functional antibody fragment on the external surface Proc Nati Acad Sci US A. 90: 10444-8 Additional methods for producing a recombinant orthogonal tRNA can be found, for example in International application publications WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" and WO 2005/019415, filed July 7, 2004. See also Forster et al., (2003) Programming peptidomimetic synthetases by translating genetic codes designed de novo PNAS 100 (11) ¡6353-6357; and, Feng et al. , (2003), Expanding tRNA recognition of a tRNA syn the tase by a single amino acid change, PNAS 100 (10): 5676-5681.

Orthogonal Aminoacyl-tRNA Synthase (O-RS) An O-RS of the present invention preferably aminoacylates an O-tRNA with an unnatural amino acid, both in vitro and in vivo. An O-RS of the invention can be provided to the translation system, for example, a cell, by a polypeptide that includes an O-RS and / or by a polynucleotide encoding an O-RS or a portion thereof. For example, an exemplary O-RS comprises an amino acid sequence as summarized in the sequence listing and examples herein, or a conservative variation thereof. In another example, an O-RS, or a portion thereof is encoded by a polynucleotide sequence encoding an amino acid comprising the sequence in the sequence listing or examples herein or a polynucleotide sequence complementary thereto. See, for example, the tables and examples herein for exemplary sequences of O-RS molecules. See also, the section entitled "Nucleic acid and polypeptide sequence and variants" herein. Methods for identifying an orthogonal aminoacyl-tRNA N synthetase (O-RS), for example an O-RS, for use with an O-tRNA, are also an aspect of the invention. For example, a method that includes subjecting, for example, positive selection, a population of cells of a first species, wherein the cells individually comprise: (1) a member of a plurality of aminoacyl-tRNA synthetases (RS), ( for example, the plurality of RS may include RS mutants, RS derived from a species different from the first species or both RS mutants and RS derived from a species different from the first species); (2) the orthogonal tRNA (O-tRNA) (e.g., of one or more species) and (3) a polynucleotide encoding a selection marker (e.g., positive) and comprising at least one selector codon. The cells are screened or filtered for those that show an improvement in suppression efficiency compared to cells lacking or with a reduced amount of the member of the plurality of RS. The Suppression efficiency can be measured by techniques knin the art and as described herein. Cells that have an improvement in suppression efficiency comprise a Active RS that aminoacylates O-tRNA. A level of aminoacylation (in vitro or in vivo) by the active RS of a first set of tRNA of the first species is compared with the aminoacylation level (in vitro or in vivo) by the active RS of a second set of tRNA of the second species. The level of ammoacylation can be determined by a detectable substance (eg, a labeled unnatural amino acid). The active RS that more efficiently ammoacylates the second set of tRNAs compared to the first set of tRNAs is commonly selected, thereby providing an efficient orthogonal ammoacyte-synthetase (optimized) for use with the O-tRNA. An O-RS, identified by the method, is also an aspect of the invention. Any of a variety of assays can be used to determine ammoacylation. These assays can be performed either in vitro or in vivo. For example, m-vitro aminoacylation analyzes are described in, for example, Hoben and Soli (1985) Methods Enzymol. 113.55-59. ammoacylation can also be determined by using a reporter together with orthogonal translation components and detecting the reporter in a cell expressing a polynucleotide comprising at least one codon selector that encodes a protein. See also, WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACID;" and WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE". The identified O-RS can be further manipulated to alter the specificity of the substrate by the smtetase, such that only an unnatural amino acid desired, but not any of the twenty common amino acids, are charged to the O-tRNA. Methods for generating an orthogonal aminoacyl-tRNA synthetase with a substrate specificity for an unnatural amino acid include mutation of the synthetase, for example in the active site in the synthetase, in the site of editing mechanism in the synthetase, in different sites at combine different domains of synthetases or the like and apply a selection process. A strategy is used that is based on the combination of a positive selection followed by a negative selection. In positive selection, the deletion of the selector codon introduced in a non-essential position (s) of a positive marker allows the cells to survive under positive selection pressure. In the presence of both natural and non-natural amino acids, the survivors thus encode active synthetases that carry the orthogonal suppressor tRNA with either a natural or a non-natural amino acid. In negative selection, the deletion of a selector codon introduced in a non-essential position (s) of a native marker removes synthetases with natural amino acid specificities. Survivors of negative and positive selection encode synthetases that aminoacylate (load) the orthogonal suppressor tRNA with non-natural amino acids only. Then these synthetases can additionally be subjected to mutagenesis, for example redistribution of RNA or other methods of recursive mutagenesis.

A library of O-RS mutants can be generated using various mutagenesis techniques known in the art. For example, mutant RSs can be generated by site-specific mutations, random point mutations, homologous recombination, DNA redistribution or other methods of recursive mutagenesis, chimeric construction or any combination thereof. For example, a library of mutant RSs can be produced from two or more of smaller, less diverse "sub-libraries." RS Chimeric Libraries are also included in the invention. It should be noted that the tRNA synthetases libraries of various organisms (e.g., microorganisms such as eubacteria or archaebacteria) such as libraries comprising natural diversity (see, e.g., U.S. Patent No. 6,238,884 issued to Short et al; 5,756,316 issued to Schallenberger et al; U.S. Patent No. 5,783,431 issued to Petersen et al; U.S. Patent No. 5,824,485 issued to Thompson et al; U.S. Patent No. 5,958,672 issued to Short et al) are optionally constructed and selected as orthogonal pairs. . Once the synthetases are subjected to the selection / positive and negative filtration strategy, these synthetases can then be subjected to additional mutagenesis. For example, a nucleic acid encoding O-RS can be isolated; a set of polynucleotides encoding mutated O-RSs (eg, by random mutagenesis, site-specific mutagenesis, recombination or any combination thereof) can be generated from the nucleic acid and these individual steps or a combination of these steps can be repeated until a mutated O-RS is obtained which preferentially aminoates the O-tRNA with the non-natural amino acid, for example an alkynyl amino acid. In an aspect of the invention, the steps are carried out multiple times, for example at least twice. Additional levels of selection / filtering severity can also be used in the methods of the invention, to produce O-tRNA, O-RS or pairs thereof. The severity of selection or filtration can be varied in one or both stages of the method to produce an O-RS. This could include, for example, varying the amount of selection / filtering agent that is used, etc. You can also make additional rounds of positive and / or negative selections. The selection or filtration may also be one or more of a change in amino acid permeability, a change in translation efficiency, change in translation fidelity, etc. Commonly, the one or more changes are based on a mutation in one or more genes in an organism in which a pair of orthogonal tRNAARN synthetase is used to produce protein. Additional general details to produce O-RS andaltering the specificity of the synthetase substrate can be found in International Publication No. WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS"; and WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE". See also, Wang and Schultz "Expanding the Genetic Code", Angewandte Chemie Int. Ed. , 44 (1): 34-66 (2005), the content of which is incorporated by reference in its entirety.

SOURCE AND GUEST ORGANISMS The orthogonal translation components (O-tRNA and O-RS) of the invention can be derived from any organism (or combination of organisms) for use in a host translation system of any other species, with the warning of that the components of O-tARN / O-RS and the host system work orthogonally. It is not a requirement that the O-tRNA and the OR-RS of an orthogonal pair be derived from the same organism. In some aspects, the orthogonal components are derived from the genus Archaea (that is, archeabacteria) for use in a eubacterial host system. For example, the orthogonal O-tRNA may be from an Archae organism, for example an archaebacteria such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium. species NRC-I, Archaeo globus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyru pernix, Methanococcus maripaludi s, Methanopyrus kandleri, Methanosarcina mazei (Mm), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus tokodaii, Thermoplasma acidophilum, Ther oplastna volcanium, or the like, or a eubacterium, such as Escherichia coli, Thermus thermophi lus, Baci llus s tearothermphilus or the like, while the orthogonal 0-RS can be derived from an organism or combination of organisms, for example a archaebacteria, such as Methanococcus jannaschii, Methanobacterium thermoautotrophi cum, Halobacterium such as Haloferax volcanii and Halobac terium NRC-I, Archa eoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshi i, Aeuropyrum pernix, Methanococcus maripaludi s, Methanopyrus kandleri, Methanosarcina mazei, Pyrobacul um aerophi l um, Pyrococcus abyssi, Sulfolobus solfa tari cus, Sulfolobus tokodaii , Thermoplasma acidophilum, Thermoplastna vol canium or the like, or a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus or the like. In one embodiment, eukaryotic sources, e.g., plants, algae, protists, fungi, yeasts, animals (e.g., mammals, insects, arthropods, etc.), or the like can also be used as sources of O-tRNA and O -RS. The individual components of a pair of O-tRNA / O-RS they can be derivatives of the same organism or different organisms. In one embodiment, the pair of O-tRNA / O-RS is from the same organism. Alternatively, the O-tRNA and the O-RS of the O-tRNA / O-RS pair are from different organisms. The O-tRNA, O-RS or pair of O-tRNA / O-RS can be selected or filtered in vivo or in vitro and / or used in a cell, for example a eubacterial cell, to produce a polypeptide with an alkynyl amino acid . The eubacterial cell used is not limited, for example, Escherichia coli, Thermus thermophilus, Bacillus s tearothermphi lus or the like. Eubacterial cell compositions comprising translation components of the invention are also an aspect of the invention. See also, international application publication number WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE", filed on April 16, 2004, to filter O-tRNA and / or O-RS into one species for use in another species. In some aspects, the O-tRNA, O-RS or O-tRNA / O-RS pair can be selected or filtered in vivo or in vitro and / or used in a cell, for example a eukaryotic cell to produce a polypeptide with a non-natural amino acid. The eukaryotic cell used is not limited, for example, to any appropriate yeast cell, such as Saccharomyces cerevisiae (S. cerevisiae) or the like, can be used. Compositions of eukaryotic cells comprising Translation components of the invention are also an aspect of the invention. Saccharomyces cerevisiae can be chosen as a species of eukaryotic host, since this organism provides several advantages. The species is unicellular, has a generation time fast and genetically well characterized. See, for example D. Burke, et al., (2000) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. In addition, since the translation machinery of eukaryotes is highly conserved (see, for example, 1996 Translational Control, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Y. Kwok, &JT Wong, (1980), Evolutionary relationship between Halobacterium cutirubrum and eukaryotes determined by the use of aminoacyl-tRNA synthetases as phylogenetic probes, Canadian Journal of Biochemistry 58: 213-218, and (2001) The Ribosome, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), O-RS genes (for example, O-RS genes derived from RS sequences of wild-type E. coli) for the incorporation of non-natural amino acids discovered in S. Cerevi siae can be introduced into higher eukaryotic organisms (e.g., in mammalian cells) and used, in association with tRNAs cognates (see, eg, K. Sakamoto, et al., (2002) If te-specific incorporation of an unnatural amino acid into proteins in mammalian cells, Nucleic Acids Res. 30: 4692-4699, and C. Kohrer, et al., (2001), Import of amber and ochre suppressor tRNAs into mammalian cells: a general approach to whether te-specific insertion of amino acid analogues into proteins, Proc. Nati Acad. Sci. U.S. A. 98: 14310-14315) to incorporate non-natural amino acids. Although orthogonal translation systems (for example, comprising an O-RS, an O-tRNA, and an unnatural amino acid) can use cultured host cells to produce proteins that have non-natural amino acids, an orthogonal translation system is not intended. of the invention requires an intact viable host cell. For example, an orthogonal translation system can use a cell-free system in the presence of a cell extract. Of course, the use of cell-free in vitro transcription / translation systems for protein production is a well-establi technique. The application of these in vitro systems to produce proteins having non-natural amino acids using components of the orthogonal translation system described herein is also within the scope of the invention.

SELECTING CODONS Selector codes of the invention expand the genetic codon structure of the protein biosynthetic machinery. For example, a selector codon includes, for example, a single three-codon basis, a nonsense codon, such as a retention codon, for example an amber codon (UAG) or an opal codon (UGA), an unnatural codon, at least a four-base codon , a rare codon or the like. A number of selector codons can be introduced to a desired gene, for example one or more, two or more or more than three, etc. By using different codons selectors, multiple orthogonal tRNA / synthetase pairs can be used that allow the site-specific incorporation of multiple non-natural amino acids, for example including at least one alkynyl amino acid, using these different codons selectors. In one embodiment, the methods involve the use of a selector codon that is a retention codon for the incorporation of an unnatural amino acid in vivo into a cell. For example, an O-tRNA is produced that recognizes the retention codon and is aminoacylated by an O-RS with an unnatural amino acid. This O-tRNA is not recognized by host aminoacyl-tRNA synthetases that occur stably in nature. Conventional site-directed mutagenesis can be used to introduce the retention codon at the site of interest into a polynucleotide encoding a polypeptide of interest. See, for example, Sayers, J.R., et al. (1988), 5 ', 3' Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 791-802. When the O-RS, O-tRNA and the nucleic acid encoding a polypeptide of interest are combined, for example in vivo, the unnatural amino acid is incorporated in response to the retention codon to give a polypeptide containing the unnatural amino acid at the specified position. In one embodiment of the invention, the retention codon used as the selector codon is an amber codon, UAG and / or an opal codon, UGA. In one example, a genetic code in which UAG and UGA are both used as a selector codon can encode 22 amino acids while retaining the ocher senseless codon, UAA, which is the most abundant termination signal. The incorporation of active amino alkyl amino acids can be done without significant disturbance of the host cell. For example in non-eukaryotic cells, such as Escherichia coll, because the suppression efficiency for the UAG codon depends on the competition between the O-tRNA, for example, the amber suppressor tRNA, and the release factor 1 (RF1) (which binds the UAG codon and initiates the release of the growing peptide from the ribosome), the suppression effciency can be modulated for example either by increasing the level of expression of O-tRNA, for example the suppressor tRNA or when using a deficient strain of RF1. In eukaryotic cells, because the suppression efficiency for the UAG codon depends on the competition between the O-tRNA, for example the amber suppressor tRNA and a eukaryotic release factor (for example, eRF) (which binds to a retention codon and initiates the release of the growing ribosome peptide), the suppression efficiency can be modulated for example by increasing the level of expression of O-tRNA, for example, suppressor tRNA . In addition, additional compounds may also be present, for example reducing agents such as dithiothreitol (DTT). Non-natural amino acids can also be encoded with rare codons. For example, when the concentration of arginine in an in vitro protein synthesis reaction is reduced, the rare arginine codon, AGG, has proven to be efficient for the insertion of Ala by a synthetic aRNA-acylated tRNA. See, for example, Ma et al., Biochemistry, 32: 7939 (1993). In this case, the synthetic TARN competes with the tARNArg that occurs in a stable manner in nature, which exists as a minor species in Escherichia coli. In addition, some organisms do not use all triplet codons. An unassigned codon AGA in My crococcus l u teus has been used for the insertion of amino acids into an in vitro transcription / translation extract. See, for example, Kowal and Oliver, Nucí. Acid Res., 25: 4685 (1997). The components of the invention can be generated to use these rare codons in vivo. Sequencing codons can also comprise extended codons, for example codons of four or more bases, such as codons of four, five, six or more bases. Examples of four-base codons include, for example, AGGA, CUAG, UAGA, CCCU, and the like. Examples of five base codons include, for example, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. The methods of the invention include using extended codons based on frame shift suppression. Codons of four or more bases can insert for example one or multiple non-natural amino acids to the same protein. In other embodiments, the anticodon loops can decode, for example, at least one codon of four bases, at least one codon of five bases or at least one codon of six bases or more. Since there are 256 codons of four possible bases, multiple non-natural amino acids can be encoded in the same cell using a codon of four or more bases. See also, Anderson et al., (2002) Exploring the Limits of codon and Anticodon Size, Chemistry and Biology, 9: 237-244; and Magliery, (2001) Expanding the genetic code: Selection of Efficient Suppressors of Four-codons bases and Identification of "Shifty" Four-base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307: 755-769. For example, four-base codons have been used to incorporate non-natural amino acids into proteins using in vitro biosynthetic methods. See, for example, Ma et al., (1993) Biochemistry, 32: 7939; and Hohsaka et al., (1999) J. Am. Chem. Soc, 121: 34. CGGG and AGGU were used to incorporate simultaneously 2-naphthylalanine and a NBD derivative of lysine to streptavidin in vitro with two chemically acylated table displacement suppressors. See, for example, Hohsaka et al., (1999) J. Am. Chem. Soc. 121: 12194. In an in vivo study, Moore et al. examined the ability of derivatives of tARNLeu with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C), and it was found that the UAGA quadruplet can be decoded by a TARNEu with an UCUA anticodon with an efficiency of 13 to 26% with little decoding in table 0 or 1. See Moore et al., (2000) J. Mol. Biol., 298: 195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the invention, which can reduce nonsense reading and suppression of frame shift in other undesirable sites. Codices of four bases have been used as codons selectors in a variety of orthogonal systems. See, for example, WO 2005/019415; WO 2005/007870 and WO 2005/07624. See also, Wang and Schultz "Expanding the Genetic Code", Angewandte Chemie Int. Ed., 44 (1): 34-66 (2005), the content of which is incorporated by reference in its entirety. While the examples below utilize an amber selector codon, codons of four or more bases can also be used, by modifying the examples herein to include four base O-tRNAs and modified synthetases to include mutations similar to those previously described for several non-natural amino acid O-RS. For a given system, a selector codon may also include one of the three natural base codons, wherein the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that lacks tRNA that recognizes the natural three-codon base and / or a system where the three-base codon is a rare codon. Selector codes optionally include non-natural base pairs. These non-natural base pairs additionally expand the existing genetic alphabet. A base pair increases the number of triplet codons from 64 to 125. The properties of third base pairs include simple and selective base pairing, efficient enzymatic incorporation to DNA with high affinity for a polymerase and efficient prolonged primer extension after of the synthesis of the unborn natural base pair. Descriptions of unnatural base pairs that can be adapted for methods and compositions, include, for example, Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20: 177-182. See also Wu, Y., et al., (2002) J. Am. Chem. Soc. 124: 14626-14630. Other relevant publications are listed below in the present. For in vivo use, the non-natural nucleoside is a permeable membrane and is phosphorylated to form the triphosphate correspondent. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. The previous efforts of Benner and others take advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most notable example of which is the iso-C: iso-G pair. See for example, Switzer et al., (1989) J. Am. Chem. Soc, 111: 8322; and Piccirilli et al., (1990) Nature, 343: 33; Kool, (2000) Curr. Opin. Chem. Biol., 4: 602. These bases in general are poorly matched to a degree with natural bases and can not be replicated enzymatically. Kool et al. Demonstrated that hydrophobic packaging interactions between bases can replace the hydrogen bond to lead to base pair formation. See Kool, (2000) Curr. Opin. Chem. Biol., 4: 602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl. , 36, 2825. In an effort to develop a non-natural base pair that satisfies all the above requirements, Schultz, Romesberg and collaborators have systematically synthesized and studied a series of non-natural hydrophobic bases. It is found that a PICS: PICS autopar is more stable than natural base pairs and can be efficiently incorporated into DNA using the Klenow fragment of DNA polymerase I Escherichia coli (KF). See, for example, McMinn et al., (1999) J. Am. Chem. Soc, 121: 11586; and Ogawa et al., (2000) J. Am. Chem. Soc, 122: 3274. A 3MN autopar: 3MN can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, for example Ogawa et al., (2000) J. Am. Chem. Soc. 122: 8803. However, both bases act as a chain terminator for additional replication. A mutant DNA polymerase has recently been developed that can be used to replicate the PICS autopar. In addition, a 7AI autopar can be replicated. See, for example Tae et al., (2001) J. Am. Chem. Soc. 123: 7439. A new pair of metallobase, Dipic: Py has also been developed, which forms a stable pair in the Cu (II) bond. See Meggers et al., (2000) J. Am. Chem. Soc, 122: 10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the method of the invention can take advantage of this property to generate orthogonal tRNAs for them. A translation deviation system can also be used to incorporate an alkynyl amino acid into a desired polypeptide. In a translation deviation system, a large sequence is inserted into a gene but is not translated into protein. The sequence contains a structure that serves as an indication to induce the ribosome to jump over the sequence and resume the translation downstream of the insert.

NON-NATURAL AMINO ACIDS As used herein, an unnatural amino acid refers to any amino acid, modified amino acid or amino acid analogue other than selenocysteine and / or pyrrolysin and the following genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. The generic structure of an alpha-amino acid is illustrated by the formula I: I A non-natural amino acid is commonly any structure having formula I wherein the R group is any substituent different from that used in the 20 natural amino acids. See, for example, Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. Note that the non-natural amino acids of the invention may be compounds that are stably present in nature other than the above twenty alpha-amino acids. Because the non-natural amino acids of the invention commonly differ from the natural amino acids in the natural chain, the non-natural amino acids form amide bonds with other amino acids, for example natural or unnatural, in the same way in which they are formed into proteins that occur in a stable manner in nature. However, non-natural amino acids have side chain groups that distinguish them from natural amino acids. Of particular interest here are the non-natural amino acids provided in Figure 1. For example, these non-natural amino acids include but are not limited to p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1, 5-dansyl-alanine, amino acid of 7-amino-coumarin, amino acid of 7-hydroxycoumarin, nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p- cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine , 3-nitro-L-tyrosine and p-nitro-L-phenylalanine. Both of the L and D enantiomers of these non-natural amino acids find use with the invention. In addition to the non-natural amino acids in the figure 1, other non-natural amino acids can be incorporated simultaneously to a polypeptide of interest, for example, by using a second pair of appropriate 0-RS / O-tRNA in conjunction with an orthogonal pair provided by the present invention. Many such additional non-natural amino acids and appropriate orthogonal pairs are known. See references cited herein. For example, see Wang and Schultz "Expanding the Genetic Code", Angewandte Chemie Int. Ed., 44 (1): 34-66 (2005), the content of which is incorporated by reference in its entirety. In other non-natural amino acids, for example, R in formula I optionally comprises an alkyl-, aryl-, acyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, ether, borate, boronate, phospho, phosphono, phosphine, enone, imine, ester, hydroxylamine, amine and the like or any combination thereof. Other non-natural amino acids of interest include, but are not limited to, amino acids comprising a photoactivatable crosslinking agent, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups , amino acids that interact covalently or non-covalently with other molecules, photoengineered and / or photoisomerizable amino acids, biotin or amino acids that contain biotin analogue, keto-containing amino acids, glycosylated amino acids, a portion of saccharide attached to the amino acid side chain, amino acids that comprise polyethylene glycol or polyether, amino acids substituted by heavy atoms, chemically cleavable or photocleavable amino acids, amino acids with an elongated side chain in comparison with natural amino acids (eg, polyethers or long-chain hydrocarbons, for example greater than about 5, greater than about 10 carbon atoms, etc.), carbon-containing sugar-containing amino acids, amino-thio-acid-containing amino acids and amino acids that contain one or more toxic portions. In another aspect, the invention provides non-natural amino acids having the general structure illustrated by formula IV below: An unnatural amino acid having this structure is commonly any structure wherein Ri is a substituent used in one of the twenty natural amino acids (eg, tyrosine or phenylalanine) and R2 is a substituent. Thus, this type of non-natural amino acid can be seen as a natural amino acid derivative. In addition to the non-natural amino acids containing novel side chains such as the alkynyl group, the non-natural alkynyl amino acids may also optionally comprise substantially modified chain structures, for example as illustrated by the structures of formula II and III: wherein Z commonly comprises OH, NH2, SH, NH-R ', or S-R'; X and Y, which may be the same or different commonly comprise S or O, and R and R ', which are optionally the same or different, are commonly selected from the same list of constituents for the R group described above for non-amino acids. naturals that have formula I also as hydrogen. For example, the non-natural amino acids of the invention optionally comprise substitutions on the amino or carboxyl group as illustrated by the formulas II and III. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates, for example with side chains corresponding to the twenty common natural amino acids or non-natural side chains. In addition, substitutions in the carbon a optionally include L, D, or α-α-disubstituted amino acids, such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid and the like. Other structural alternatives include cyclic amino acids, such as proline analogs as well as proline analogs with 3, 4, 6, 7, 8 and 9 ring members, β and β. amino acids such as β-alanine acid and substituted β-amino butyric acid. In some aspects, the invention utilizes non-natural amino acids of L-configuration. However, it is not intended that the invention be limited to the use of non-natural amino acids of L-configuration. It is contemplated that the D-enantiomers of these non-natural amino acids will also find use with the invention. Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines and meta-substituted tyrosines, wherein the substituted tyrosine comprises an alkynyl group, an acetyl group, a benzoyl group, amino group, hydrazine, hydroxyamine, a thiol group, carboxy group, isopropyl group, methyl group, a straight or branched chain hydrocarbon of C6-C20 a saturated or unsaturated hydrocarbon, an O-methyl group, polyether group, nitro group or the like. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs of the invention include, but are not limited to, α-hydroxy derivatives, β-substituted derivatives, cyclic derivatives and derivatives of amino acid glutamine substituted. Exemplary phenylalanine analogues include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines and meta-substituted phenylalanines, wherein the substituent comprises an alkynyl group, a hydroxy group, a methoxy group, a methyl group, a group allyl, an aldehyde, a nitro, a thiol group, a keto group or the like. Specific examples of non-natural amino acids include, but are not limited to, p-ethylthiocarbionyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1,5-dansyl-alanine, amino acid of 7-amino-coumarin, amino acid of 7-hydroxycou-arine, nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine , 3-amino-L-tyrosine, bipyridyl alanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine and p-nitro-L- phenylalanine Also, a p-propargiloxyphenylalanine, a 3-dihydroxy-L-phenylalanine (DHP), a 3,4,6-trihydroxy-L-phenylalanine, a 3,4,5-trihydroxy-L-phenylalanine, 4-nitro- phenylalanine, a p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, an L-3 - (2 -naphthyl) alanine, a 3-methyl-phenylalanine, a 0-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a 3-nitro-tyrosine, a 3-thiol-tyrosine, a tri-O-acetyl-GlcNAcß-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p- benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, and an isopropyl-L-phenylalanine and the like. Structures of a variety of non-natural amino acids are provided herein, see for example figure 1. See also, published international application WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE".

Chemical synthesis of non-natural amino acids Many of the non-natural amino acids provided above are commercially available, for example from Sigma (USA) or Aldrich (Milwaukee, WI, USA). Those that are not commercially available are optionally synthesized as stipulated in various publications or using standard methods known to those of skill in the art. For organic synthesis techniques, see for example Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of non-natural amino acids include for example WO 2002/085923 entitled "In vivo Incorporation of Unnatural Amino Acids"; Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King, F.E. & Kidd, D.A.A. (1949) A New Synthesis of Glutamine and of? -Dipeptides of Glutamic Acid from Phthylated Intermediates. J. Chem. Soc, 3315-3319; Friedman, O.M. & Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig, J.C. et al. (1988) Absolute Configuration of Enantiomers of 7-Chloro-4 [[4- (diethylamino) -1-methylbutyl] amino] quinoline (Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991) Glutamine analogues as Potential Antimalarials,. Eur. J. Med. Chem. 26, 201-5; Koskinen, A.M.P. & Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B.D. & Rapoport, H. (1985) Synthesis of Optically Puré Pipecolates from L-Asparagine. Application to the Total Synthesis of (+) - Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization J. Org. Chem. 1989: 1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of L- and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43: 4297-4308; and Subasinghe et al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site. J. Med. Chem. 35: 4602-7. See also international publication WO 2004/058946, entitled "PROTEIN ARRAYS", filed on December 22, 2003.

Cellular Absorption of Non-natural Amino Acids The absorption of non-natural amino acid by a cell is a matter that is commonly considered when designing and selecting non-natural amino acids, for example for incorporation into a protein. For example, the high charge density of the α-amino acids suggests that these compounds are unlikely to be permeable to the cell. Natural amino acids are absorbed into the cell via a collection of protein-based transport systems that frequently exhibit various degrees of amino acid specificity. A quick selection can be made that determines which non-natural amino acids, if any, are absorbed by the cells. See for example toxicity analyzes for example in the international publication WO 2004/058946, entitled "PROTEIN ARRAYS", filed on December 22, 2003; and Liu and Schultz (1999) Progress towards the evolution of an ogranism with an expanded genetic code. PNAS 96: 4780-4785. Although absorption is easily analyzed with several analyzes, an alternative to designing non-natural amino acids that are prone to cellular absorption pathways is to provide biosynthetic pathways to create amino acids in vivo.

Biosynthesis of non-natural amino acids There are already many biosynthetic pathways in cells for the production of amino acids and other compounds, whereas a biosynthetic method for a particular non-natural amino acid may not exist in nature, for example in a cell, the invention provides such methods. For example, biosynthetic pathways for non-natural amino acids are optionally generated in host cells by adding new enzymes or modifying existing host cell pathways. Additional novel enzymes are enzymes that are optionally presented in a stable manner in nature or artificially evolved enzymes. For example, the biosynthesis of p-aminophenylalanine (as presented in an example in WO 2002/085923, supra) depends on the addition of a combination of known enzymes from other organisms. The genes for these enzymes can be introduced into a cell by transforming the cell with a plasmid comprising the genes. Genes, when expressed in the cell, provide an enzymatic route to synthesize the desired compound. Examples of enzyme types that are optionally added are provided in the examples below. Sequences of additional enzymes are found, for example in Genbank. Artificially developed enzymes are also optionally added to a cell in the same way. In this way, the machinery and cellular resources of a cell are manipulated to produce non-natural amino acids. Of course, any of a variety of methods can be used to produce new enzymes for use in biosynthetic pathways or for evolution of existing routes, for the production of non-natural amino acids, in vitro or in vivo. Many commercially available methods for the development of enzymes and other biosynthetic pathway components can be applied to the present invention to produce non-natural amino acids (or of course, to evolve synthetases to have new substrate specificities or other activities of interest). For example, DNA redistribution is optionally used to develop new enzymes and / or routes of such enzymes for the production of non-natural amino acids (or production of new synthetases), in vitro or in vivo. See for example Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370 (4): 389-391; and Stemmer, (1994), DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Nati Acad. Sci. USA., 91: 10747-10751. A related procedure redistributes families of related genes (eg homologues) to rapidly develop enzymes with desired characteristics. An example of such methods of "family gene redistribution" is found in Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature, 391 (6664): 288-291. New enzymes (either biosynthetic pathway components or synthetases) can also be generated using a DNA recombination procedure known as "incremental truncation for the creation of hybrid enzymes" ("ITCHY"), for example, as described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology" Nature Biotech 17: 1205. This method can also be used to generate an enzyme library and other route variants that can serve as substrates for one or more of in vitro or in vivo recombination methods. See also Ostermeier et al. (1999) "Combinatorial Protein Engineering by Incremental Truncation", Proc. Nati Acad. Sci. USA, 96: 3562-67, and Ostermeier et al. (1999), "Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts," Biological and Medicinal Chemistry, 7: 2139-44. Another method uses exponential assembly mutagenesis to produce enzyme libraries and other pathway variants that are, for example, selected for the ability to catalyze a relevant biosynthetic reaction to produce an unnatural amino acid (or a novel synthetase). In this procedure, small groups of residues in a sequence of interest are randomized in parallel to identify, in each altered position, amino acids that lead to functional proteins. Examples of such methods, which can be adapted to the present invention to produce new enzymes for production of non-natural amino acids (or new smtetases) are found at Delegrave & Youvan (1993) Biotechnology Research 11: 1548-1552. In yet another procedure, random or semi-random mutagenesis using doped or degenerate oligonucleotides for the enzyme design and / or as a pathway can be used, for example by using the general mutagenesis methods of Arkm and Youvan (1992) "Optimizmg nucleotide mixtures to encode specific subsets of ammo acids for semi-random mutagenesis "Biotechnology 10297-300, or Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods Enzymol. 208,564-86 Still another procedure, often termed "non-stochastic" mutagenesis, which utilizes polymucleotide reassembly and saturation mutagenesis at the site can be used to produce enzymes and / or pathway components, which can then be selected as soon as possible. to the ability to carry out one or more functions of smtetase or biostatic route (for example, for the production of non-natural amino acids m alive). See, for example Short "NON-STOCHASTIC GENERATION OF GENETIC VACCINES AND ENZIMES" WO 00/46344. An alternative to such mutational methods involves the recombination of whole genomes of organisms and resulting progeny selection for particular path functions (often referred to as "redistribution of whole genome.) This method can be applied to the present invention, for example by genomic recombination and selection of an organism (eg, an E. coli or another cell) in terms of the ability to produce an unnatural amino acid (or For example, the methods taught in the following publications can be applied to the route design for the evolution of existing routes and / or new routes in cells to produce unnatural amino acids in vivo: Patnaik et al. (2002) "Genome shuffling of lactobacillus for improved acid tolerance" Nature Biotechnology 20 (7): 707-712; and Zhang et al. (2002) "Genome shuffling leads to rapid phenotypic improvement in bacteria" Nature, February 7, 415 (6872) : 644-646 Other techniques for the design of organisms and metabolic pathway design, for example, for the production of desired compounds are also available and can also be applied to the production of amino acids. Unnatural uses Examples of publications that teach useful route design procedures include: Nakamura and White (2003) "Metabolic engineering for the microbial production of 1,3 propanediol" Curr. Opin. Biotechnol. 14 (5): 454-9; Berry et al. (2002) "Application of Metabolic Engineering to improve both the production and use of Indigo Biotech" J. Industrial Microbilogy and Biotechnology 28: 127-133; Banta et al. (2002) "Optimizing an artificial metabolic pathway: Engineering the co-factor specificity of Corynebacterium 2, 5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis "Biochemistry 41 (20), 6226-35; Selivonova et al. (2001)" Rapid Evolution of Novel Traits in Microorganisms "Applied and Environmental Microbiology, 67: 3645, and many others.Without consideration of the method used, commonly, the amino acid not noted produced with a biosynthetic route designed of the invention is produced in a sufficient concentration for efficient protein biosynthesis, for example a cellular amount natural, but not to such a degree as to significantly affect the concentration of other cellular amino acids or to deplete cellular resources, typical concentrations produced in vivo in this manner are from about 10 mM to about 0.05 mM Once a cell is designed to produce desired enzymes for a specific pathway and an unnatural amino acid is generated, selections in vivo are optionally used for op further optimize the production of the non-natural amino acid for ribosomal protein synthesis and cell growth.

Orthogonal components for incorporating non-natural amino acids The invention provides compositions and methods for producing orthogonal components to incorporate non-natural amino acids, for example the non-natural amino acids provided in Figure 1, to a growing polypeptide chain in response to a selector codon, eg, an amber stop codon, a nonsense codon, a codon of four or more bases, e.g. in vivo For example, the invention provides orthogonal tRNAs (O-tRNA), orthogonal aminoacyl-tRNA N synthetases (O-RS) and pairs thereof. These pairs can be used to incorporate an unnatural amino acid into growing polypeptide chains. A composition of the invention includes an orthogonal aminoacyl-tRNA N synthetase (O-RS), wherein the O-RS preferably aminoacylates an O-tRNA with p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine , 1,5-dansyl-alanine, 7-amino-coumarin alanine, 7-hydroxy-coumarin alanine, o-nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p -cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine; p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine or p-nitro-L-phenylalanine. In certain embodiments, the O-RS comprises an amino acid sequence comprising any of SEQ ID NOS: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59-63 and conservative variations thereof. In certain embodiments of the invention, O-RS preferably aminoacylates O-tRNA over any endogenous tRNA with an unnatural amino acid. particular, wherein the O-RS has predisposition by the O-tRNA and wherein the proportion of O-tRNA loaded with an unnatural amino acid to the endogenous tRNA loaded with the same unnatural amino acid is greater than 1: 1 and more preferably in where the O-RS charges the O-tARN exclusively or almost exclusively. A composition that includes an O-RS may optionally also include an orthogonal tRNA (O-tRNA), wherein the 0-tRNA recognizes a selector codon. Commonly, an O-tRNA of the invention includes at least about, for example, a suppression efficiency of 45%, 50%, 60%, 75%, 80%, or 90% or more in the presence of a cognate smtetase in response to a selector codon, compared to the deletion efficiency of an O-tRNA that comprises or is encoded by a sequence of polynucleotides as summarized in the sequence listings (e.g., SEQ ID NO: 1) and examples herein In one embodiment, the efficiency of suppression of O-RS and O-tRNA is for example 5 times, 10 times, 15 times, 20 times, 25 times or more greater than the efficiency of suppression of O-tRNA in the absence of an O-RS. In some aspects, the efficiency of suppression of the O-RS and the O-tRNA together is at least 45% of the efficiency of suppression of an orthogonal tyrosyl-tARN smtetase pair derived from Methanococcus annaschu or alternatively, a pair of leukocyte orthogonal tRNA synthetase derived from E. coli. A composition that includes an O-tRNA may include optionally a cell (e.g., a eubacterial cell, such as an E. coli cell and the like or a eucapone cell such as a yeast cell), and / or a translation system. A cell (e.g., a eubacterial cell or a yeast cell) comprising a translation system is also provided by the invention, wherein the translation system includes an orthogonal tRNA (O-tRNA); an ammoacyl-tRNA orthogonal synthetase (O-RS); and an unnatural amino acid, for example an amino acid shown in Figure 1. Commonly, O-RS preferably ammoacylates O-tRNA with respect to any endogenous tRNA with the non-natural amino acid, where O-RS is predisposed by the O-tRNA, and wherein the proportion of O-tRNA charged with the unnatural amino acid to the endogenous tRNA loaded with the unnatural amino acid is greater than 1-1 and more preferably wherein the O-RS charges the exclusive O-tRNA or Almost exclusively. The O-tRNA recognizes the first selector codon and the O-RS preferably ammoacilates the O-tRNA with a non-natural amino acid. In one embodiment, the O-tRNA comprises or is encoded by a sequence of polynucleotides as summarized in SEQ ID NO: 1, SEQ ID NO: 2, or a sequence of polynucleotides complementary thereto. In one embodiment, the O-RS comprises an amino acid sequence as summarized in any of SEQ ID NOS: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57, 59-63, and conservative variations thereof. A cell of the invention can optionally further comprise a different pair of additional O-tRNA / O-RS and a second unnatural amino acid, for example where this O-tRNA recognizes a second selector codon and this O-RS is preferably ammonia O-RS. -tRNA corresponding to the second non-natural amino acid, wherein the second amino acid is different from the first non-natural amino acid. Optionally, a cell of the invention includes a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the O-tRNA. In certain embodiments, a cell of the invention is a eubacterial cell (such as E. coll) or a yeast cell, which includes an orthogonal tRNA (O-tRNA), an ammoacyl-tRNA orthogonal (O-RS), a unnatural amino acid and a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises the selector codon that is recognized by the O-tRNA. In certain embodiments of the invention, O-RS preferably ammoacylates O-tRNA with the unnatural amino acid with an efficiency that is greater than the efficiency at which O-RS aminoacylates any endogenous tRNA. In certain embodiments of the invention, an O-tAR? of the invention comprises or is encoded by a sequence of polynucleotides as summarized in the sequence listings (eg, SEQ ID NO: 1 or SEQ ID NO: 2) or examples herein, or a polynucleotide sequence complementary thereto. In certain embodiments of the invention, an O-RS comprises an amino acid sequence as summarized in the sequence listings or a conservative variation thereof. In one embodiment, an O-RS or a portion thereof is encoded by a polynucleotide sequence encoding an amino acid as summarized in the sequence listings or examples herein, or a polynucleotide sequence complementary thereto. The O-tRNA and / or the O-RS of the invention can be derived from any of a variety of organisms (e.g., eukaryotic and / or non-eukaryotic organisms). Polynucleotides are also an aspect of the invention. A polynucleotide of the invention includes an artificial polynucleotide (eg, artificial and not occurring in a stable manner in nature) comprising a nucleotide sequence encoding a polypeptide as summarized in the sequence listings herein and / or is complementary to that sequence of polynucleotides. A polynucleotide of the invention may also include a nucleic acid that hybridizes to a polynucleotide described above, under highly stringent conditions, over substantially the entire length of the nucleic acid. A The polynucleotide of the invention also includes a polynucleotide that is, for example, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical to that of a tRNA. which is stably present in the corresponding coding nature or nucleic acid (however a polynucleotide of the invention is different than a tRNA that is stably present in nature or a corresponding coding nucleic acid) where, the tRNA recognizes a selector codon, for example, a four-base codon. Artificial polynucleotides which are for example at least 80%, at least 90%, at least 95%, at least 98% or more identical to any of the foregoing and / or a polynucleotide comprising a conservative variation of any of the above are also included in the polynucleotides of the invention. Vectors comprising a polynucleotide of the invention are also an aspect of the invention. For example, a vector of the invention may include a plasmid, a cosmid, a virus, a vector of the invention and / or the like. A cell comprising a vector of the invention is also an aspect of the invention. Methods for producing components of a pair of 0-tRNA / 0-RS are also aspects of the invention. The components produced by these are also aspects of the invention.

For example, methods for producing at least one tRNA that is orthogonal to a cell (O-tRNA) include generating a tAR library? mutants; mutate an anticodon loop from each member of the tAR library? mutants to allow recognition of a selector codon, thereby providing an O-tAR library? potential and subject to negative selection a first population of cells of a first species, wherein the cells comprise a member of the library of O-tAR? potential The negative selection removes cells that comprise a member of the O-tAR library? potentials that is aminoacylated by an aminoacylAR? synthetase (RS) that is endogenous to the cell. This provides a cluster of tAR? which are orthogonal to the cell of the first species, thereby providing at least one O-tAR ?. An O-tAR? produced by the method of the invention is also provided. In certain embodiments, the methods further comprise subjecting a second population of cells of the first species to positive selection, wherein the cells comprise a member of the tAR? that are orthogonal to the cell of the first species, an aminoacyl -tar? Cognate synthetase and a positive selection marker. When using positive selection, cells are selected or filtered for those cells that comprise a member of the tAR? which are aminoacylated by the aminoacyl-tAR? cognata synthetase and that shows a desired response in the presence of the positive selection marker, thereby providing an O-tRNA. In certain embodiments, the second cell population comprises cells that were not eliminated by the negative selection. Methods for identifying an aminoacylAR are also provided? orthogonal synthetase that charges an O-tAR? with an alkynyl amino acid. For example, the methods include subjecting a population of cells from a first spice to a selection, wherein each of the cells comprises: (1) a member of a plurality of aminoacylAR? synthetases (RS), (for example, the plurality of RSs may include RS mutants, RSs derived from a different species of a first species or both RS mutants and RSs derived from a species different from the first species); (2) the tAR? orthogonal (O-tAR?) (for example, of one or more species) and (3) a polynucleotide that encodes a positive selection marker and comprises at least one selector codon. Cells (e.g., a host cell) are screened or filtered on those that show an improvement in suppression efficiency compared to cells lacking or having a reduced amount of the member of the plurality of RS. These selected / filtered comprise an active RS that aminoacilates the O-tAR ?. An aminoacyl -tar? Orthogonal synthetase identified by the method is also an aspect of the invention.

Methods for producing a protein in a cell (e.g., in a eubacterial cell such as an E. coli cell or the like or in a yeast cell) having the non-natural amino acid in a selected position are also an aspect of the invention . For example, a method involves culturing, in an appropriate medium, a cell, wherein the cell comprises a nucleic acid comprising at least one selector codon and encodes a protein, providing the unnatural amino acid and incorporating the unnatural amino acid into the position specified in the protein during translation of the nucleic acid with at least one selector codon, thereby producing the protein. The cell further comprises: an orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon and an orthogonal aminoacyl-tRNA N synthetase (O-RS) that preferentially aminoacylates O-tRNA with the unnatural amino acid. A protein produced by this method is also an aspect of the invention. The invention also provides compositions that include proteins, wherein the proteins comprise, for example, p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1,5-dansyl-alanine, amino acid of 7- amino-coumarin, amino acid of 7-hydroxycoumarin, nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L- phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p- (2- amino-1-hydroxyethyl) -L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine or p-nitro-L-phenylalanine. In certain embodiments, the protein comprises an amino acid sequence that is at least 75% identical to that of a known protein, for example, a therapeutic protein, a diagnostic protein, an industrial enzyme, or portion thereof. Optionally, the composition comprises a pharmaceutically acceptable carrier.

NUCLEIC ACID AND POLYPEPTIDE SEQUENCE AND VARIANTS As described herein, the invention provides polynucleotide sequences encoding, for example, O-tRNA and O-RS, and polypeptide amino acid sequences, for example, O-RS and for example , compositions, systems and methods comprising the polynucleotide or polypeptide sequences. Examples of the sequences, for example amino acid and nucleotide sequences of O-tRNA and O-RS are disclosed herein (see Table 5, for example, SEQ ID NOS: 7-10, 12, 14, 16, 18, 20 , 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59-63). However, one skilled in the art will appreciate that the invention is not limited to those sequences disclosed herein, for example, in the examples and sequence listings. The one skilled in the art will appreciate that the invention also provides many sequences related to the functions described herein, for example polynucleotides and polypeptides that encode conservative variants of an O-RS disclosed herein. The construction and analysis of orthogonal synthetase species (O-RS) which are capable of aminoacylating an O-tRNA with an unnatural amino acid, for example, a non-natural amino acid provided in Figure 1, are described in Examples 1 to 16 These examples describe the construction and analysis of O-RS species that are able to incorporate the unnatural amino acids p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1,5-dansyl-alanine , 7-amino-coumarin-alanine, 7-hydroxy-coumarin-alanine, o-nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, -cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine; p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine and p-nitro-L-phenylalanine. The invention provides polypeptides (O-RS) and polynucleotides, for example O-tRNA, polynucleotides encoding O-RS or portions thereof, oligonucleotides used to isolate aminoacyl-tRNA synthetase clones, etc. The polynucleotides of the invention include those that encode proteins or polypeptides of interest of the invention with one or more codons selectors. In addition, the polynucleotides of the invention include, for example, a polynucleotide comprising a nucleotide sequence as summarized in SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 and 58, and a polynucleotide that is complementary to or that encodes a polynucleotide sequence thereof. A polynucleotide of the invention also includes any polynucleotide encoding a 0-RS amino acid sequence comprising SEQ ID NO: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 , 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59-63. Similarly, an artificial nucleic acid that hybridizes to a polynucleotide indicated above under highly stringent conditions over substantially the entire length of the nucleic acid (and is different from a polynucleotide that occurs stably in nature) is a polynucleotide of the invention. In one embodiment, a composition includes a polypeptide of the invention and an excipient (e.g., pH buffer, water, pharmaceutically acceptable excipient, etc.). The invention also provides an antibody or antiserum specifically immunoreactive with a polypeptide of the invention. An artificial polynucleotide is a polynucleotide that is artificial and does not occur in a stable manner in nature. A polynucleotide of the invention also includes an artificial polynucleotide that is, for example, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical to that of a tRNA that is presented Stably in nature (but it is different from a tRNA that occurs in a stable way in nature). A polynucleotide also includes an artificial polynucleotide that is, for example, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical (but not 100% identical) to that of a tRNA that is presented in a stable manner in nature. In certain embodiments, a vector (eg, a plasmid, a cosmid, a phage, a virus, etc.) comprises a polynucleotide of the invention. In one embodiment, the vector is an expression vector. In another embodiment, the expression vector includes a promoter operably linked to one or more of the polynucleotides of the invention. In another embodiment, a cell comprises a vector that includes a polynucleotide of the invention. Those skilled in the art will also appreciate that many variants of the disclosed sequences are included in the invention. For example, conservative variants of the disclosed sequences that produce a functionally identical sequence are included in the invention. Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence, are considered to be included in the invention. Unique subsequences of the sequences disclosed herein, as determined for example, standard comparison sequence techniques, are also included in the invention.

Conservative variants Due to the degenerof the genetic code, "silent substitutions" (that is, substitutions in a nucleic acid sequence that do not result in an alteration in a coded polypeptide) are an implicated aspect of each nucleic acid sequence encoding a nucleic acid sequence. Similarly, "conservative amino acid substitutions", wherein one or a limited number of amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also easily identified as being highly similar to a developed construct. Such conservative variations of each disclosed sequence are an aspect of the present invention "Conservative variants" of a particular nucleic acid sequence refers to those nucleic acids that encode identical or essentially identical amino acid sequences or wherein the nucleic acid does not encode an amino acid sequence, to sequences essentially identical. Those skilled in the art will recognize that individual substitutions, cancellations or additions that alter, add or cancel a single amino acid or a small percentage of amino acids (commonly less than 5%, more commonly less than 4%, 2% or 1%) in a coded sequence are "conservatively modified variations" wherein the alterations result in the cancellation of an amino acid, addition of an amino acid or substitution of an amino acid with a chemically similar amino acid. Thus, "conservative variants" of a listed polypeptide sequence of the present invention includes substitutions of a small percentage, commonly less than 5%, commonly less than 2% or 1%, of the amino acids of the polypeptide sequence, with a amino acid from the same conservative substitution group. Finally, the addition of sequences that do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional sequence, is a conservative variation of the basic nucleic acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art, wherein an amino acid residue is replaced by another amino acid residue having similar chemical properties (eg, aromatic side chains or positively charged side chains) and consequently does not substantially change the functional properties of the polypeptide chain. The following summarizes exemplary groups containing natural amino acids of similar properties, wherein substitutions within a group is a "conservative substitution".

TABLE 1 Nucleic Acid Hybridization Comparative hybridization can be used to identify nucleic acids of the invention, in which conservative nucleic acid variants of the invention are included and this method of comparative hybridization is a preferred method for distinguishing nucleic acids of the invention. In addition, target nucleic acids that hybridize to a nucleic acid represented by SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 and 58, under conditions of high, ultra-high and ultra-ultra high severity are one aspect of the invention. Examples of such nucleic acids include those with one or fewer silent or conservative nucleic acid substitutions compared to a given nucleic acid sequence. It is said that a test nucleic acid hybridizes specifically to a probe nucleic acid when it hybridizes at least 50% also as to the probe to the perfectly corresponding complementary target, that is, with a signal-to-noise ratio at least half as high as the probe's hybridization to the target under conditions in which the perfectly corresponding probe is linked to the perfectly corresponding complementary target with a signal-to-noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the non-corresponding target nucleic acids. Nucleic acids "hybridize" when they are associated, commonly in solution. Nucleic acids are hybridized due to a variety of well-characterized physicochemical strengths, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide for nucleic acid hybridization is found in Tijssen (1993) Labora tory Techniques in Biochemi s try and Biology Molecular- Hybridi zation wi th Nuclei c Acid Probes part I chapter 2, "Overview of principies of hybridization and the strategy of nucleic acid probé assays," (Elsevier, New York), also as in Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented through 2004) ("Ausubel"); Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgms 1) and Hames and Higgms (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, Engly (Hames and Higgms 2) provides details regarding the synthesis, labeling, detection and quantification of DNA and RNA in which oligonucleotides are included. An example of severe hybridization conditions for the hybridization of complementary nucleic acids having more than 100 complementary residues on a filter in a Southern blot or northern blot is 50% formalma with 1 mg of hepapna at 42 ° C, the hybridization is carried performed all night long An example of severe washing conditions is a 0.2x SSC wash at 65 ° C for 15 minutes. { see, Sambrook, supra for a description of the pH regulator solution of SSC). Frequently, high severity washing is preceded by a low severity wash to remove the background probe signal. An example of low severity wash SSC 2x at 40 ° C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed by an unrelated probe in the particular hybridization analysis indicates detection of a specific hybridization. "Severe hybridization washing conditions" in the context of nucleic acid hybridization experiment such as Southern and northern hybridizations are sequence dependent and are different or different environmental parameters. An extensive guide regarding the hybridization of Nucleic acids are found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Severe hybridization and washing conditions can easily be determined empirically for any test nucleic acid. For example, in the determination of severe hybridization and washing conditions, the conditions of hybridization and washing are gradually increased (for example, by increasing the temperature, decreasing the salt concentration, increasing the concentration of detergent and / or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria is met. For example, under highly stringent hybridization and washing conditions, the hybridization and washing conditions are gradually increased until a probe is linked to a perfectly corresponding complementary target with a signal-to-noise ratio that is at least 5x as high as that observed for the hybridization of the probe to an uncorrected target. "Very severe" conditions are selected to be equal to the thermal melting point (Tm) for a particular probe. The Tm is the temperature (under ionic strength and defined pH) at which 50% of the test sequence is hybridized to a perfectly corresponding probe. For the purposes of the present invention, in general, "highly severe" hybridization and washing conditions are selected to be approximately 5 ° C lower than the Tm for the specific sequence at a defined ionic strength and pH. Hybridization and washing conditions of "Ultra-high severity" are those in which the severity of the hybridization and washing conditions are increased until the ratio of signal to noise for the linkage of the probe to the perfectly corresponding complementary target nucleic acid is at least 10x as high as that observed for hybridization to any of the target nucleic acids without corresponding. An objective nucleic acid that hybridizes to a probe under such conditions, with a signal-to-noise ratio of at least 1/2 that of the perfectly corresponding complementary target nucleic acid is said to bind to the probe under ultrasound conditions. high severity Similarly, even higher levels of severity can be determined by gradually increasing the hybridization and / or washing conditions of the relevant hybridization analysis. For example, those in which the severity of the hybridization and washing conditions are increased until the ratio of signal to noise for the linkage of the probe to the perfectly corresponding complementary target nucleic acid is at least 10X, 20X, 50X, 100X , or 500X or more as high as that observed for hybridization to any of the target nucleic acids without correspond. An objective nucleic acid that hybridizes to a probe under such conditions, with a signal-to-noise ratio of at least 1/2 of the perfectly corresponding complementary target nucleic acid is said to bind to the probe under conditions of ultra-ultra-high severity . Nucleic acids that do not hybridize to each other under severe conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code.

Unique subsequences In some aspects, the invention provides a nucleic acid comprising a unique subsequence in a nucleic acid selected from the sequences of O-tRNA and O-RS disclosed herein. The unique subsequence is unique in comparison to a nucleic acid corresponding to any known O-tRNA or O-RS nucleic acid sequence. The alignment can be performed using for example BLAST adjusted to predetermined parameters. Any single subsequence is useful, for example, as a probe to identify the nucleic acids of the invention. Similarly, the invention includes a polypeptide that it comprises a single subsequence in a polypeptide selected from the O-RS sequences disclosed herein. Here, the unique subsequence is unique in comparison to a polypeptide corresponding to any of the known polypeptide sequence. The invention also provides nucleic acids that hybridize under severe conditions to a single coding oligonucleotide that encodes a single subsequence in a polypeptide selected from the O-RS sequences, wherein the unique subsequence is unique compared to a polypeptide corresponding to any of control polypeptides (e.g., original sequences of which the synthetases of the invention were derived, e.g., by mutation). The unique sequences are determined as indicated above.

Comparison of sequence, identity and homology The terms "identical" or "percent identity", in the context of two or more nucleic acid sequences or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described later in the present (or other algorithms available to people of skill) or by visual inspection. The phrase "identical substance" in the context of two nucleic acids or polypeptides (e.g., DNA encoding an O-tRNA or O-RS or the amino acid sequence of an O-RS) refers to two or more sequences or subsequences. having at least about 60%, about 80%, about 90-95%, about 98%, about 99% or more of identity of nucleotide or amino acid residues, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. It is considered that such "substantially identical" sequences are "homologous" without reference to the actual ancestors. Preferably, the "substantial identity" exists over a region of the sequences that is at least about 50 residues in length, more preferably in a region of at least about 100 residues and more preferably, the sequences are substantially identical with respect to at least about 150 residues or over the full length of the two sequences to be compared. Proteins and / or protein sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologs when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. For example, any nucleic acid that occurs stably in nature can be modified by any appropriate mutagenesis method to include one or more codons selectors. When expressed, this mutagenized nucleic acid encodes a polypeptide comprising one or more non-natural amino acids. The mutation process can of course, additionally alter one or more standard codons, thereby changing one or more standard amino acids in the resulting mutant protein as well. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of sequence similarity that is useful for establishing homology varies with the nucleic acid and protein in question, but as little as 25% sequence similarity is used systematically to establish homology. Higher levels of sequence similarity, for example 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining percentages of sequence similarity (eg, BLASTP and BLASTN using predetermined parameters) are described herein and are generally available. For the comparison of sequence and determination of homology, commonly the sequence acts as a reference sequence to which the test sequences are compared. When a sequence comparison algorithm is used, the test and reference sequences are input to a computer, subsequence coordinates are designated, if necessary, and the program parameters of the sequence algorithm are designated. Then the sequence comparison algorithm calculates the percent sequence identity for the test sequence (s) in relation to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be effected, for example by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by searching by the Pearson & amp; Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see, in general, Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc., supplemented through 2004).

An example of an algorithm that is appropriate for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215: 403-410 (1990). Programming elements to perform BLAST analyzes are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high-scoring sequence pairs (HSP) by identifying short words of length W in the interrogation sequence that either matches or satisfies the positive value threshold score T when it is aligned to a word of the same. length in a database sequence. T is referred to as the neighbor word score threshold (Altschul et al., Supra). These initial neighbor word scores act as seeds for initial searches to find HP longer than they contain. Word scores are then issued in both directions along each song sequence to the cumulative alignment score can be increased. Cumulative scores are calculating, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always> 0) and N (punishment score for residues that do not match, always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of Word annotations in each annotation are stopped when: the cumulative alignment score falls by the amount X of its maximum obtained value; the cumulative score goes to zero or less, due to the accumulation of one or more negative-residue residue alignments or the end of either one sequence or another is reached. The parameters of the BLAST algorithm W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, a hope (E) of 10, a cut of 100, M = 5, N = -4, and a comparison of both strands , for amino acid sequences, the BLASTP program uses a word length (W) of 3 as a default, a hope (E) of 10, and the BLOSUM62 score matrix (see Henikoff &Henikoff (1989) Proc. Nati. Sci. USA 89: 10915). In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin &Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873 -5787 (1993)). A measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of probability by which a match between two nucleotide or amino acid sequences would occur by probability, for example, an acid nucleic is considered similar to a reference sequence if the smallest sum probability in a comparison of test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and more preferably less than about 0.001.

Mutagenesis and other molecular biology techniques The polynucleotides and polypeptides of the invention and used in the invention can be manipulated using molecular biology techniques. General texts describing molecular biology techniques include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001 ("Sambrook") and Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds. , Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2004) ("Ausubel"). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics concerning, for example, the generation of genes that include codons selectors for the production of proteins that include alkynyl amino acids (for example pPRO-Phe), orthogonal tRNAs, orthogonal synthetases, and pairs of them.

Various types of mutagenesis are used in the invention, for example to mutate tRNA molecules, to produce tRNA libraries, to produce libraries of synthetases, to insert selector codons that encode an alkynyl amino acid into a protein or polypeptide of interest. They include but are not limited to site-directed random point mutagenesis, homologous recombination, redistribution of AD? or other methods of recursive mutagenesis, chimeric construction, mutagenesis using templates containing uracil, oligonucleotide-directed mutagenesis, mutagenesis of AD? modified by phosphorothioate, mutagenesis using AD? duplex with spaces or the like or any combination thereof. Additional appropriate methods include point mismatch repair, mutagenesis using deficient repair host strains, restriction-selection and restriction-purification, cancellation mutagenesis, mutagenesis by total gene synthesis, double-strand rupture repair and the like. Mutagenesis, for example involving chimeric constructs, is also included in the present invention. In one embodiment, mutagenesis can be guided by known information of molecules that occur stably in nature or molecules that occur stably in nature altered or modulated, eg, sequence, sequence comparisons, properties physical, crystalline structure or the like. The host cells are genetically engineered (eg, transformed, transduced or transfected) with the polynucleotides of the invention or constructs that include a polynucleotide of the invention, for example a vector of the invention, which can be for example a cloning vector or an expression vector. For example, the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase and the protein to be derived are operably linked to gene expression control elements that are functional in relation to the desired host. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences and promoters useful for the regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences that allow replication of the cassette in eukaryotes or prokaryotes or both (eg, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. The vectors are suitable for replication and / or integration in prokaryotes, eukaryotes or preferably both. See Giliman & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328: 731 (1987); Schneider, B., et al., Protein Expr. Purif. 6435: 10 (1995); Ausubel, Sambrook, Berger (all above). He vector can for example be in the form of a plasmid, a bacterium, a virus, a naked polynucleotide or a conjugated polynucleotide. The vectors are introduced into cells and / or microorganisms by standard methods in which electroporation is included (From et al., Proc.Nat.Acid.Sci.USA 82, 5824 (1985), infection by viral vectors, ballistic penetration of high speed by small particles with the nucleic acid either within the matrix of beads or small particles or on the surface (Klein et al., Nature 327, 70-73 (1987)), and / or the like. a single highly efficient and versatile plasmid was developed for site-specific incorporation of non-natural amino acids into proteins in response to the amber retention codon (UAG) in E. coli.In the new system, the pair of tARNtyr (CUA) suppressor M jannaschi i and tyrosyl-tRNA synthetase are encoded in a single plasmid, which is compatible with most E. coli expression vectors The monokistronic tRNA operon under the control of the proK promoter and terminator was constructed for optimal secondary structure and processing of tRNA. The introduction of a mutated form of the glnS promoter for the synthetase resulted in a significant increase in both suppression efficiency and fidelity. Increases in suppression efficiency were also obtained by multiple copies of the tRNA gene as well as by a specific mutation (D286R) in the synthetase (Kobayashi et al., "Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion", Nat. Struct. Biol., 10 (6): 425-432 [2003]). The generality of the optimized system was also demonstrated by highly efficient and exact incorporation of several different non-natural amino acids, whose unique utilities in the study of protein function and structure were previously tested. A catalog of bacteria and bacteriophages useful for cloning is provided, for example by the ATCC, for example, The ATCC Catalog of Bacteria and Bacteriophage (1996) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Sambrook (supra), Ausubel (supra), and Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be adapted or ordered standard from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX mcrc.com ), The Great American Gene Company (Ramona, CA available on the web at genco.com), ExpressGen Inc. (Chicago, IL available on the web at expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others .

The engineered host cells can be cultured in modified conventional nutrient media as appropriate for such activities such as for example selection steps, promoter activation or selection of transformants. These cells can optionally be cultured in transgenic organisms. Other useful references, for example for isolation and cell culture (for example, for isolation of subsequent nucleic acids) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

PROTEINS AND POLYPEPTIDES OF INTEREST Methods for producing a protein in a cell with an alkynyl amino acid at a specified position are also an aspect of the invention. For example, a method includes culturing, in an appropriate medium, the cell, wherein the cell comprises a nucleic acid comprising at least one selector codon and encoding a protein and providing the alkynyl amino acid; wherein the cell further comprises: an orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon and an aminoacyl-tAR? orthogonal synthetase (O-RS) which preferably aminoacylates O-tRNA with the alkynyl amino acid. A protein produced by this method is also an aspect of the invention. In certain embodiments, the O-RS comprises a predisposition for the aminoacylation of O-tAR? Cognate with respect to any TAR? endogenous in an expression system. The relative proportion between O-tAR? and TAR? endogenous that is loaded by the O-RS when the O-tAR? and O-RS are present in molar or equal concentrations, is greater than 1: 1, preferably by at least about 2: 1, more preferably 5: 1, still more preferably 10: 1, still more preferably 20: 1, still more preferably 50: 1, still more preferably 75: 1, still more preferably 95: 1, 98: 1, 99: 1, 100: 1, 500: 1, 1,000: 1, 5,000: 1 or higher. The invention also provides compositions that include proteins, wherein the proteins comprise an alkynyl amino acid. In certain embodiments, the protein comprises an amino acid sequence that is at least 75% identical to that of a therapeutic protein, a diagnostic protein, an industrial enzyme or portion thereof. The compositions of the invention and compositions made by the methods of the invention optionally they are in a cell. The O-tRNA / O-RS pairs or individual components of the invention can then be used in a translation machinery of the host system, which results in an alkynyl amino acid that is incorporated into a protein. The international publications numbers WO 2004/094593, filed on April 16, 2004, entitled "EXPA? DI? G THE EUKARYOTIC GE? ETIC CODE," and WO 2002/085923, entitled "I? VIVO I? CORPORATIO? OF U? ? ATURAL AMI? O ACIDS ", describe this process and are incorporated herein by reference. For example, when a pair of O-tAR? / O-RS is introduced to a host, for example an Escheri chia coli cell or a yeast cell, the pair leads to the in vivo incorporation of a non-natural amino acid such as p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenyl-alanine, 1,5-dansyl-alanine, 7-amino-coumarin alanine, 7-hydroxy-coumarin alanine, o-nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine; p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine or p-nitro-L-phenylalanine to a protein in response to a selector codon. The non-natural amino acid that is added to the system can be a synthetic amino acid, such as a derivative of a phenylalanine or tyrosine, which can be added exogenously to the culture medium.

Optionally, the compositions of the present invention may be in an in vitro translation system or in an in vivo system (s). A cell of the invention provides the ability to synthesize proteins comprising unnatural amino acids in large useful amounts. In one aspect, the composition optionally includes, for example 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams or more of the protein comprising an alkynyl amino acid or an amount that can be obtained with protein production methods in vivo (details on when to recombinant protein production and purification are provided herein). In another aspect, the protein is optionally present in the composition at a concentration of for example, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, per at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more in for example, a cell lysate, a pH buffer solution, a pharmaceutical pH buffer solution or other liquid suspension (for example, in a volume of, for example, any of about 1 nL to about 100 L). The production of large amounts (for example, greater than that common possible with other methods, for example in vitro translation) of a protein in a cell includes at least one alkynyl amino acid is an aspect of the invention. The incorporation of a non-natural amino acid can be done to, for example, adapt changes in the structure and / or function of protein, for example to change the size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of target sites of protease, targeting a portion (for example for a protein arrangement), incorporation of labels or reactive groups, etc. Proteins that include an unnatural amino acid may have improved or even completely new catalytic or physical properties. For example, the following properties are optionally modified by including a non-natural amino acid to a protein: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic ability, half-life (e.g. serum), ability to react with other cells, for example covalently or non-covalently and the like.

The compositions include proteins that include at least one unnatural amino acid are useful for, for example, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (eg, antibodies) and for example the study of structure and function of protein. See, for example, Dougherty, (2000) Amino Acids as Probes of Pro tein Structure and Function, Current Opinion in Chemical Biology, 4: 645-652. In some aspects of the invention, a composition includes at least one protein with at least one, for example at least two, at least three, at least four, at least five, at least six, so minus seven, at least eight, at least nine, or at least ten or more non-natural amino acids. The non-natural amino acids may be the same or different, for example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different sites in the proteins comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different non-natural amino acids. In another aspect, a composition includes a protein with at least one, but less than all, of a particular amino acid present in the protein is an unnatural amino acid. For a given protein with more than one non-natural amino acid, the non-natural amino acids may be identical or different (for example, the protein may include two or more different types of non-natural amino acids, or may include two thereof). non-natural amino acid). For a given protein with more than two non-natural amino acids, the non-natural amino acids may be the same, different or a combination of a multiple non-natural amino acid of the same kind with at least one different unnatural amino acid. Essentially any protein (or portion thereof) that includes an alkynyl amino acid (and any corresponding coding nucleic acid, which includes one or more selector codons) can be produced using the compositions and methods herein. No attempt is made to identify the hundreds of thousands of known proteins, any of which can be modified to include one or more non-natural amino acids, for example by adaptation of any available mutation methods to include one or more appropriate codons selectors in a relevant translation system. Common sequence repertoires for known proteins include GenBank EMBL, DDBJ and the NCBI. Other repertoires can be easily identified by searching the internet. Commonly, the proteins are for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to any available protein (eg, a therapeutic protein, a diagnostic protein, an industrial enzyme, or portion thereof and the like) and they comprise one or more non-natural amino acids. Examples of therapeutic proteins, diagnostics and other proteins that can be modified to comprise one or more non-natural amino acids can be found, but not limited to, those in international publications WO 2004/094593, filed on April 16, 2004, entitled "Expanding the Eukaryotic Genetic Code", and WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS". Examples of therapeutic, diagnostic proteins and other proteins that can be modified to comprise one or more non-natural amino acids include, but are not limited to, for example alpha-1 antitrypsin, angiostatma, antihemolytic factor, antibodies (further details regarding antibodies are found later herein), apolipoprotein, apoprotein, atrial natropytic factor, atpal natriuretic polypeptide, atrial peptides, CXC chemokines (eg, T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), calcitonin, CC chemokines (e.g., monocyte chemoattractant protein-1, monocyte chemoattractant protein-2) , monocyte protein-3 protein, monocyte inflammatory protein-1 alpha, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40 ligand, C-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokines (eg, peptide-78 epithelial neutrophil activator, GROa / MGSA, GROß, GRO ?, MlP-la, MlP-ld, MCP-1), epidermal growth factor (EGF), erythropoietin ("EPO"), toxins A and B exfoliators, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growth factors, Hedgehog proteins ( for example, sonic, Indian, desert), hemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumin, insulin, insulin-like growth factor (IGF), interferons (eg, IFN-a, IFN -β, IFN-?), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12, etc.), keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibiting factor (NIF), oncostatin M, osteogenic protein, hormone parathyroid, PD-ECSF, PDGF, peptide hormones (eg, human growth hormone), pleiotropin, protein A, protein G, pyrogenic exotoxins A, B and C, relaxin, rennin, SCF, soluble complement receptor, soluble I-CAM 1, receptors of soluble interleukin (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, this es, staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), superoxide dismutase (SOD), syndrome toxin of toxic shock (TSST-1), thymosin alfa 1, tissue plasminogen activator, tumor necrosis vein factor (TNF beta), tumor necrosis factor receptor (TNFR), tumor necrosis factor-alpha (TNF) alpha), vascular endothelial growth factor (VEGEF), urokinase and many others A class of proteins that can be made using the compositions and methods for in vivo incorporation of alkynyl amino acids described herein includes transcriptional modulators or a portion thereof. Exemplary transcriptional modulators include genes and transcriptional modulator proteins that modulate cell growth, differentiation, regulation or the like. Transcriptional modulators are found in prokaryotes, viruses and eukaryotes, which include fungi, plants, yeasts, insects and animals that include mammals that provide a wide range of therapeutic targets. It will be appreciated that expression and transcription activators regulate transcription through many mechanisms, for example by binding to receptors, stimulation of a cascade of signal transduction, regulation of expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind to promoters and enhancers, unwinding of DNA, splicing of pre - mRNA, polyadenylation of RNA and degradation of AR ?. A class of proteins of the invention (for example, proteins with one or more alkynyl amino acids) include biologically active proteins such as cytokines, inflammatory molecules, growth factors, their receptors, and oncogenic products, for example, interleukins (eg, IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF / c-Kit, CD40L / CD40, VLA-4 / VCAM- 1, ICAM-1 / LFA-1, and hialaurin / CD44; signal translation molecules and corresponding oncogene products, for example, Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, for example, p53, Tat, Fos, Myc, Jun, Myb, Rei, and steroid hormone receptors such as those for estrogen, progesterone, testosterone, aldosterone, the LDL receptor ligand and corticosterone. Enzymes (eg, industrial enzymes) or portions thereof with at least one alkynyl amino acid are also provided by the invention. Examples of enzymes include, but are not limited to, for example, amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transaerases, haloperoxidases, monooxygenases (e.g., p450), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases.

Many of these proteins are commercially available (see, for example, the Sigma BioSciences 2002 catalog and price list) and the corresponding protein sequences and genes and commonly, many variants thereof are well known (see, for example, Genbank). Any of them can be modified by the insertion of one or more alkyl amino acid according to the invention, for example to alter the protein with respect to one or more therapeutic, diagnostic or enzymatic properties of interest. Examples of therapeutically relevant properties include serum half-life, storage half-life, stability, immunogenicity, therapeutic activity, detectability (e.g., by inclusion of reporter groups (e.g., tags or label binding site) at the amino acids unnatural), LD50 reduction or other side effects, ability to enter the body through the gastric system (eg, oral availability) or the like Examples of diagnostic properties include serum half-life, stability, diagnostic activity, detectability or the like. Examples of relevant enzymatic properties include average storage life, stability, enzymatic activity, production capacity or the like. A variety of other proteins may also be modifications to include one or more alkyl amino acids using compositions and methods of the invention. For example, the invention may include substitution of one or more natural amino acids in one or more vaccine proteins with an alkynyl amino acid, for example in infectious fungal proteins, for example Aspergillus species, Candida; bacteria, particularly E. coli, which serves as a model for pathogenic bacteria, also as medically important bacteria such as Staphylococci (for example, aureus) or Streptococci (for example, pneumoniae); protozoa such as sporozoa (for example, Plasmodia), rhi zopodos (for example, In tamoeba) and flagellates. { Trypanosoma, Lei shmania, Tri chomonas, Giardia, etc.); viruses such as virus (+) RNA (examples include Poxvirus for example, vaccinia; Picornavirus, eg polio; Togavirus, eg, rubella; Flavivirus, eg, HCV; and Coronavirus); (-) RNA (eg, Rhabdoviruses, eg, VSV, paramixovimus, eg, RSV, Ortomixovimses, eg influenza, Bunyavirus and Arenavirus), dsADA virus (Reovirus, for example), RNA virus to DNA, that is, retroviruses, e.g., HIV and HTLV, and certain DNA-to-RNA viruses such as Hepatitis B. Agriculturally-related proteins such as insect-resistant proteins (e.g., Cry proteins), starch and lipid production enzymes , toxins from plants and insects, toxin resistance proteins, Mycotoxin detoxification proteins, plant growth enzymes (eg, Ribose 1, 5-bisphosphate carboxylase / oxygenase, "RUBISCO"), lipoxygenase (LOX), and phosphoeolpyruvate (PEP) carboxylase are also appropriate targets for modification of alkynyl amino acid In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and / or compositions of the invention is encoded by a nucleic acid. Commonly, the nucleic acid comprises at least one selector codon, at least two codons selectors, at least three codons selectors, at least codon selectors box, at least five codons selectors, at least six codons selectors, so minus seven codons selectors, at least eight codons selectors, at least nine codons selectors, ten or more codons selectors. Genes coding for proteins or polypeptide of interest can be mutagenized using methods well known to those of skill in the art and described herein under "Mutagenesis and other molecular biological techniques" to include, for example, one or more codons selectors for the incorporation of a non-natural amino acid. For example, a nucleic acid for a protein of interest is mutagenized to include one or more codons selectors, providing the insertion of one or more non-natural amino acids. The invention includes any such variants, for example, mutants, versions of any protein, for example that include at least one non-natural amino acid. Similarly, the invention also includes corresponding nucleic acids, that is any nucleic acid with one or more codons selectors that encodes one or more non-natural amino acids. To make a protein that includes an unnatural amino acid, host cells and organisms that are adapted for in vivo incorporation of the non-natural amino acid via orthogonal tRNA / RS pairs can be used. The host cells are genetically engineered (eg, transformed, transduced or transfected) with one or more vectors expressing the orthogonal tRNA, the orthogonal tRNA synthetase and a vector encoding the protein to be derived. Each of these components may be in the same vector or may be in a separate vector or two components may be in one vector and the third component in a second vector. The vector may for example be in the form of a plasmid, a bacterium, a virus, a naked polynucleotide or a conjugated polynucleotide.

Definition of polypeptides by immunoreactivity Because the polypeptides of the invention provide a variety of novel polypeptide sequences (for example, polypeptides comprising alkynyl amino acids in the case of proteins synthesized in the systems of In the present or for example, in the case of new synthetases, new sequences of standard amino acids), the polypeptides also provide new structural aspects that can be recognized, for example in immunological tests. The generation of antisera, which bind specifically to the polypeptides of the invention, also as polypeptides that are linked by such antisera, is an aspect of the invention. The term "antibody", as used herein, includes but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes or fragments thereof that specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric antibodies and single chain antibodies and the like. Fragments of immunoglobulins in which Fab fragments are included and fragments produced by an expression library, in which phage display are included, are also included in the term "antibody" as used herein. See, for example, Paul, Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, for antibody structure and terminology. In order to produce antisera for use in an immunoassay or immunological test, one or more of the immunogenic polypeptides is produced and purified as described herein. For example, it can be produced recombinant protein in a recombinant cell. An inbred strain of mice (used in this analysis because the results are more reproducible due to the virtual genetic identity of the mice) are immunized with the immunogenic protein (s) in combination with a standard adjuvant, such as Freund's adjuvant and a standard mouse immunization protocol (see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, test formats immunological and conditions that can be used to determine the specific immunoreactivity Additional data regarding proteins, antibodies, antisera, etc. can be found in the international publications WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2002 / 085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACID", WO 2004/035605, entitled "GLYCOPROTEIN SYNTHESIS" and WO 2004/05 8946, entitled "PROTEIN ARRAYS".

USE OF O-TARN, O-RS AND PAIRS OF O-TARN / O-RS The compositions of the invention and compositions made by the methods of the invention are optionally in a cell. The O-tRNA / O-RS pairs or individual components of the invention can then be used in a translation machinery of the host system, which results in an alkynyl amino acid that is incorporated into a protein. International publication No. WO 2002/085923 to Schultz, et al., Entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS," describes this process and is incorporated herein by reference. For example, when a pair of O-tRNA / O-RS is introduced to a host, for example Escheri chia coli or yeast, the pair conducts the in vivo incorporation of an unnatural amino acid, which can be added exogenously to the culture medium. , to a protein, for example a myoglobin test protein or a therapeutic protein, in response to a selector codon, for example an amber non-sense codon. Optionally, the compositions of the invention may be in an in vitro translation system or in a cellular in vivo system (s). Proteins with the non-natural amino acid can be used in any of a wide range of applications. For example, the unnatural portion incorporated into a protein can serve as an objective for any of a wide range of modifications. For example, cross-linking with other proteins, with small molecules such as labels or dyes and / or biomolecules. With these modifications, the incorporation of the non-natural amino acid can result in improved therapeutic proteins and can be used to alter or improve the catalytic function of enzymes. In some aspects, the incorporation and subsequent modification of a non-natural amino acid in a protein can facilitate studies in terms of protein structure, interactions with other proteins and the like.

PHOTOGRAPHY AND PHOTOENJAULMENT Photoregulated aminoacids (for example photochromic, photo-dopable, photoisomerizable, etc.) can be used to spatially and temporally control a variety of biological processes, for example, by directly regulating the activity of enzymes, receptors, ion channels or similar or by modulating the intracellular concentrations of several signaling molecules. See for example Shigeri et al., Pharmacol. Therapeut. , 2001, 91:85; Curley, et al., Pharmacol. Therapeut., 1999, 82: 347; Curley, et al., Curr. Op. Chem. Bio., 1999, 3:84; "Caged Compounds" Methods in Enzymology, Marriott, G., Ed, Academic Press, NY, 1998, V. 291; Adams, et al., Annu. Rev. Physiol. , 1993, 55: 755+; and Bochet, et al., J. Chem. Soc., Perkin 1, 2002, 125. In various embodiments herein, the compositions and methods comprise photoregulated amino acids. For example, the invention provides orthogonal translation systems for the incorporation of the non-natural photoregulated amino acids 0-nitrobenzyl-serine and O- (2-nitrobenzyl) -L-tyrosine (see, Figure 1 and Examples 8 and 9). "Photoregulated amino acids" are commonly, for example, photosensitive amino acids. The amino acids Photoregulates are in general those that are controlled in some way by light (for example UV, IR, etc.). Thus, for example, if a photoregulated amino acid is incorporated into a polypeptide having biological activity, the illumination can alter the amino acid, thereby changing the biological activity of the peptide. Some photoregulated amino acids may comprise "photoenaged amino acids", "photosensitive amino acids", "photolabile amino acids", "photoisomerizable amino acids", etc. "Caged species" such as caged amino acids or caged peptides, are those trapped inside a larger entity (eg molecule) and that are released after specific illumination. See, for example, Adams, et al., Annu. Rev. Physiol., 1993, 55: 755-784. "Ajaculating" amino acid groups can inhibit or hide (for example, by breaking bonds that would usually stabilize interactions with target molecules by changing the hydrophobicity or ionic character of a particular side chain or by steric hindrance, etc.) the biological activity in a molecule , for example a peptide comprising such an amino acid. "Photoisomerizable" amino acids can change isomeric forms due to exposure to light. The different isomers of such amino acids can end up having different interactions with other side chains in a protein after incorporation. The photoregulated amino acids can thus regulate biological activity 8 and either by means of activation, partial activation, deactivation, partial deactivation, modified activation, etc.) of the peptides in which they are present. See Adams referred to above and other references in this section for definitions and additional examples of amino acids and photo-regulated molecules. A number of photoregulated amino acids are known in the art and many are commercially available. Methods for attaching and / or associating photoregulatory portions to amino acids are also known. Such photoregulated amino acids are generally prone to various embodiments herein. It will be appreciated that while a number of possible photoregulatory portions, for example photoenzynt groups and the like, also as a number of photoregulated amino acids are listed in the one presented, such an appointment or should be interpreted as limiting. Thus, the present invention is also prone to photoregulatory and photoregulated amino acid portions that are not specifically cited herein. As stated, a number of methods are optionally applicable to create a photoregulated amino acid. Thus, for example, a photoregulated amino acid, for example a photoengineered amino acid, can be created by protecting its alpha-amino group with compounds such as BOC (butyloxycarbonyl), and protecting the α-carboxyl group with compounds such as a t-butyl ester. . Such protection can be followed by reaction of the amino acid side chain with a photolabile cage group such as 2-nitrobenzyl, in a reactive form such as 2-nitrobenzylchloroformate, a methyl ester of a-carboxyl-2-nitrobenzyl bromide or 2-nitrobenzyl diazoethane. After the caged photolabile group is added, the protective groups can be removed via standard procedures. See, for example, USPN 5,998,580. As another example, lysine residues can be caged using 2-nitrobenzylchloroformate to derive the e-amino-amino group thereby eliminating the positive charge. Alternatively, lysine can be caged by introducing a negative charge to a peptide (having such lysine) by using a cage group of a-carboxy 2-nitrobenzyloxycarbonyl. Additionally, phosphoserine and phosphothreonine can be caged by treating the phosphoamino acid or the phosphopeptide with 1 (2-nitrophenyl) diazoethane. See, for example, Walker et al., Meth Enzymol. 172: 288-301, 1989. A number of other amino acids are also easily prone to standard caging chemistry, for example serine, threonine, histidine, glutamine, asparagine, aspartic acid and glutamic acid. See, for example, Wilcox et al., J. Org. Chem. 55: 1585-1589, 1990). Again, it will be appreciated that the appointment of particular photoregulated amino acids (amino acids and / or those capable of being converted to photoregulated forms) should not be taken necessarily as limiting. Amino acid residues can also be photoregulated (for example, photosensitive or photolabile) in other ways. For example, certain amino acid residues may be created wherein the irradiation causes cleavage of a fundamental chain of peptide at the particular amino acid residue. For example, a photolabile glycine, 2-nitrophenyl glycine, can function in such a manner. See, for example, Davis, et al., 1973, J. Med. Chem., 16: 1043-1045. The irradiation of peptides having 2-nitrophenylglycine will cleave the fundamental chain of the peptide between the alpha carbon and the alpha amino group of 2-nitrophenylglycine. Such a cleavage strategy is generally applicable to amino acids other than glycine, if the 2-nitrobenzyl group is inserted between the alpha carbon and the alpha amino group. A large number of photoregulatory groups, for example, cage groups and a number of reactive compounds used to covalently attach such groups to other molecules such as amino acids, are well known in the art. Examples of photoregulatory groups (eg, photolabiles, cages) include, but are not limited to: o-nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, nitroindolines; N-acyl-7-nitroindolines; phenacyls; hydroxyphenacyl; Brominated 7-hydroxycoumarin-4-ylmethyl (e.g., Bhc); benzoin esters; dimethoxybenzoin; goal- phenols; 2-nitrobenzyl; 1- (4,5-dimethoxy-2-nitrophenyl) ethyl (DMNPE); 4, 5-dimethoxy-2-nitrobenzyl (DMNB); alpha-carboxy-2-nitrobenzyl (CNB); 1- (2-nitrophenyl) ethyl (NPE); 5-carboxymethoxy-2-nitrobenzyl (CMNB); (5-carboxymethoxy-2-nitrobenzyl) oxy) carbonyl; (4,5-dimethoxy-2-nitrobenzyl) oxy) carbonyl; Deoxybenzoinyl; and the like. See, for example, USPN 5,635,608 to Haugland and Gee (June 3, 1997) entitled "a-carboxy caged compounds" Neuro 19, 465 (1997); J Physiol 508.3, 801 (1998); Proc Nati Acad Sci USA 1988 Sep, 85 (17): 6571-5; J Biol Chem 1997 Feb 14, 272 (7): 4172-8; Neuron , 619-624, 1998; Nature Genetics, vol. 28: 2001: 317-325; Nature, vol. 392.1998: 936-941; Pan, P., and Bayley, H. "Caged cystein and thiophosphoryl peptides" FEBS Letters 405: 81-85 (1997); Pettit et al. (1997) "Chemical two-photon uncaging: a novel approach to mapping glutamate receptors" Neuron 19: 465-471; Furuta et al. (1999) "Brominated 7-hydroxycoumarin-4 -ylmethyls: novel photolabile protecting groups with biologically useful cross-sections for two photon photolysis" Proc. Nati Acad. Sci. 96 (4): 1193-1200; Zou et al. "Catalytic subunit of protein kinase A caged at the activating phosphotreonine" J. Amer. Chem. Soc. (2002) 124: 8220-8229; Zou et al. "Caged Thiophosphotyrosine Peptides" Angew. Chem. Int. Ed. (2001) 40: 3049-3051; Conrad II et al. "p-Hydroxyphenacyl Phototriggers: The reactive Excited State of Phosphate Photorelease" J. Am. Chem. Soc. (2000) 122: 9346-9347; Conrad II et al. "New Phototriggers 10: Extending the p, p * Absorption to Relay Peptides in Biological Media" Org. Lett. (2000) 2: 1545-1547; Givens et al. "A New Phototriggers 9: p-Hydroxyphenacyl as a C-Termmus Photoremovable Protecting Group for Oligopeptides" J. Am. Chem. Soc. (2000) 122: 2687-2697, Bishop et al. "40-Am? Nomet? L-2, 20-b? Pyr? Dyl-4-carboxyl? C acid (Abe) and Related Derivatives: Novel Bipyridine Ammo Acids for the Solid-Phase Incorporation of a Metal Coordination Site Withm a Peptide Backbone "Tetrahedron (2000) 56: 4629-4638; Chmg et al. "Polymers As Surface-Based Tethers with Photolytic tnggers Enablmg Laser- Induced Release / Desorption of Covalently Bound Molecules" Bioconjugal Chemi s try (1996) 7.525-8, BioPiobes Handbook, 2002 from Molecular Probes, Inc., and Handbook of Fluorescent Probes and Research Products, Ninth Edition or Web Edition, from Molecular Probes, Inc., as well as the references cited therein. Many compounds, equipment, etc. for use in the anchoring of several molecules are commercially available, for example from Molecular Probes, Inc. Additional references are found, for example, in Memfield, Science 232: 341 (1986) and Come, JET and Trentham, DR (1993) In: Biological Applications of Photochemical Switches, ed., Morrison, H., John Wiley and Sons, Inc. New York, pp. 243-305. Examples of groups in appropriate photosensitive aulators include, but are not limited to, 2-n? Trobenc? Lo, benzoin esters, N-ac? L-7-n? T? Ndolmas, meta-phenols, and fenacyls. In some embodiments, a photoregulator group (eg, cage) may optionally comprise a first linker portion, which may be linked to a second linker portion. For example, a commercially available caged phosphoramidite [1-N- (4, 4 ' -methoxytptyl) -5- (6-biotmamidocaproamidomethyl) -1- (2-mtrofeml) -ethyl] -2-cyanoethyl- (N, N-dnsopropyl) -phosphoramidite (PC Biotin Phosphoramadite, from Glen Research Corp., www.glenres .com) comprises a photolabile group and a biotome (the first binding portion). A second binding portion, for example streptavidin or avidma, can thus be linked to the caging group, increasing its volume and its effectiveness in the caking. In certain embodiments, a caged component comprises two or more caging groups each comprising a first binding portion. and the second binding portion can bind two or more first binding portions, simultaneously. For example, the caged component can comprise at least two biotylated cage groups, in streptavidin binding to multiple biotage portions on multiple caged components molecules binds the caged components to a large network. The cleavage of the photolabile group that attaches the biotome to the component results in the dissociation of the network. Traditional methods to create polypeptides caged (in which, for example, peptide substrates and proteins such as antibodies or transcription factors) are included, for example by reacting a polypeptide with a caging compound or by incorporation of a caged amino acid during synthesis of a polypeptide. See, for example, US Patent No. ,998,580 issued to Fay et al. (December 7, 1999) entitled "Photosensitive caged macromolecules"; Kossel et al. (2001) PNAS 98: 14702-14707; Trends Plant Sci (1999) 4: 330-334; PNAS (1998) 95: 1568-1573; J. Am. Chem. Soc. (2002) 124: 8220-8229; Pharmacology & Therapeutics (2001) 91: 85-92; and Angew. Chem. Int. Ed. Engl. (2001) 40: 3049-3051. A photolabile polypeptide linker (e.g., for connection of a protein transcription domain and a sensor or the like) may for example comprise a photolabile amino acid such as that described in U.S. Patent No. 5,998,580. The irradiation with light can for example release a side chain residue of an amino acid that is important for the activity of the polypeptide comprising such an amino acid. additionally, in some embodiments, the non-caged amino acids can cleave the peptide's fundamental chain from the peptide comprising the amino acid and can thus, for example, open a cyclic peptide to a linear peptide with different biological properties, etc. Activation of a caged peptide can be done by means of destruction of a photosensitive caging group on a photoregulated amino acid by any standard method known to those skilled in the art. For example, a photosensitive amino acid can be de-entrained or activated by exposure to an appropriate conventional light source, such as lasers (e.g., emitting in the UV or infrared range). Those of skill in the art will be aware and familiar with a number of appropriate lasers of wavelength and appropriate energies also as appropriate application protocols (eg exposure duration, etc.) which are applicable for use with photoregulated amino acids such as those used in the present. The release of photoregulated caged amino acids allows the control of the peptides comprising such amino acids. Such control can be both in terms of location and in terms of time. For example, focused laser exposure can de-link amino acids at one site, while not unlinking amino acids at other sites. Those skilled in the art will appreciate a variety of assays that can be used to evaluate the activity of a photoregulated amino acid, for example the assays described in the examples herein. A wide range of eg cellular function, tissue function, etc. they can be analyzed before and after the introduction of a peptide comprising an amino acid photoregulated to the cell or tissue, also as after the release of the photoregulated molecule. The compositions and methods herein can be used in a number of aspects, for example, photoregulated amino acids (e.g., in peptide) can deliver therapeutic compositions to discrete sites of a body, since the release or activation / deactivation / etc . of the photoregulated amino acid can be localized through the exposure of targeted light, etc. It will also be appreciated that the methods, structures and compositions of the invention are applicable for incorporation / use of photoregulated natural amino acids (eg those with photoregulatory portions appended / associated therewith). Photochromic and photocleavable groups can be used to spatially and temporally control a variety of biological processes, either by directly regulating the activity of enzymes (see, for example Westmark, et al., J. Am. Chem. Soc. 1993, 115: 3416-19 and Hohsaka, et al., J. A. Chem. Soc. 1994, 116: 413-4), receptors (see, for example, Bartels, et al., Proc. Nati. Acad. Sci. USA, 1971, 68: 1820-3; Lester, et al., Nature 1977, 266: 373-4: Cruz, et al., J. Am. Chem. Soc, 2000, 122: 8777-8; and Pollitt, et al. ., Angew, Chem. Int. Ed. Engl., 1998, 37: 2104-7), or ion channels (see, for example, Lien, et al., J. Am. Chem. Soc. 1996, 118: 12222-3; Borisenko, et al., J. Am. Chem. Soc. 2000, 122: 6364-70; and Banghart, et al., Nat. Neurosci. 2004, 7: 1381-6.), Or by modulating the intracellular concentrations of several signaling molecules (see, for example, Adams, et al., Annu, Rev. Physiol., 1993, 55: 755-84). In general, this requires the chemical modification of either a protein or small molecule with a photoreactive ligand such as azobenzene or a nitrobenzyl group. The ability to genetically incorporate photosensitive amino acids into proteins at defined sites directly in living organisms would significantly extend the scope of this technique. See, for example, Wu, et al., J. Am. Chem. Soc. 2004, 126: 14306-7.

EQUIPMENT The equipment is also an aspect of the invention. For example, a kit for producing a protein comprising at least one alkynyl amino acid in a cell is provided, wherein the kit includes a container containing a polynucleotide sequence encoding an O-tRNA and / or an O-tRNA and / or a polynucleotide sequence encoding an O-RS, and / or an O-RS. In one embodiment, the kit further includes a non-natural amino acid such as p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin alanine. , 7-hydroxy- coumarin alanine, o-nitrobenzyl-serine, 0- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino -L-tyrosine, bipyridylalanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine; p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine or p-nitro-L-phenylalanine. In another embodiment, the kit further comprises instructional materials for producing the protein.

EXAMPLES The following examples are offered to illustrate but not to limit the claimed invention. That of skill will recognize a variety of non-critical parameters that can be altered without deviating from the scope of the claimed invention.

EXAMPLE 1 Orthogonal translation components for the in vivo incorporation of 3-nitro-L-tyrosine into proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of 3-nitro-L-tyrosine (see, figure 1) to proteins using E. coli cell translation machinery. New orthogonal tRNA / synthetase pairs derived from M. jannaschii were isolated that function in a host cell system of E. coli.

New orthogonal synthetases were derived from tyrosyl tRNA synthetase from M. jannaschii and were used in conjunction with the tyrosyl-tARNCuA suppressed from M. jannaschii (SEQ ID NO: 1). These new orthogonal pairs have no affinity or have very low affinity for any of the common amino acids (this is, which are presented in a stable way in nature). The derived orthogonal synthetases tRNAs selectively load tyrosyl-tARNCuA amber suppressor with 3-nitro-L-tyrosine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate 3-nitro-L-tyrosine in response to an amber TAG retention codon (a selector codon). ) found in a transcript. The orthognathicity of these tRNA / synthetase pairs ensures that neither tRNA nor synthetases cross-react with endogenous E. coli tRNAs or synthetases and that the unnatural amino acid is only administered in response to TAG. The new synthetases were isolated using protocols previously described, see, for example, Alfonta et al., Journal of the American Chemical Society 125 (48): 14662-14663 (2003); and international publication WO 2005/038002, published on April 28, 2005. A library of tyrosyl-tRNA-synthetase from M. jannaschii mutants was generated by mutagenesis of the wild-type tyrosyl-tAR? -synthase M. jannaschii. The amino acid and polynucleotide sequences of the tyrosyl-tRNA-synthetase molecule of wild-type M. jannaschii are shown in Table 5 and provided in SEQ ID NOS: 3 and 4, respectively. The mutagenesis consisted of allocating active site residues predicted based on the crystal structure of the homologous tyrosyl tRNA-smtetase from Baci llus s tearo thermophi 1 us. Following the mutagenesis, the cluster of smtetases in the mutant library was passed through five rounds of positive and negative selection. This selection produced seven smtetase clones that had the ability to load the O-tRNA with 3-nitro-L-tyrosma, denoted as clones A to G. These selected smtetase clones were sequenced and their amino acid sequences were determined, as follows . Table 2 All clones A, B, C, E and G converged to the same mutant sequence. Clones D and F showed different sequences. The amino acid sequences of these mutant synthetases are provided in Table 5, SEQ ID NOs: 7-9). EXAMPLE 2 Orthogonal translation components for the in vivo incorporation of p-nitro-L-phenylalanine (N02-Phe) into proteins in E. coli This example describes the genetically programmed, site-specific incorporation of p-nitro-L-phenylalanine ( see, figure 1, also written as N02-Phe) to proteins in E. coli using a new orthogonal translation system. The unnatural amino acid N02-Phe has been used as a photoaffinity labeling probe to study the structure of the protein receptor (Dong, Mol, Pharmacol, 2005, 69, 1892), and as a fluorescence quencher for investigating protease activity ( Wang, Biochem, Biophys, Res. Corran, 1994, 201: 835) and protein structure (Sisido, J. Am. Chem. Soc. 1998, 120: 7520, 2002, 124: 14586). This amino acid has been specifically incorporated into the site to proteins with an in vitro biosynthetic method using three-base codons (Schultz, Science 1989, 244: 182), four bases (M. Sisido) and five bases (M. Sisido, Nucleic Acids Res. 2001,29, 3646). However, this procedure commonly produces only small amounts of protein.

In addition, the method is limited due to the need for stoichiometric amounts of acylated tRNA and the inability to regenerate the aminoacyl-tRNA. In view of these limitations, a new in vivo orthogonal translation system was developed to incorporate the unnatural amino acid directly into proteins, as described hereinafter. To genetically encode? 02-Phe in E. coli, the specificity of a tyrosyl-tAR? Methanococcus jannaschi i orthogonal synthetase (MjTyrRS) was altered in such a way that the synthetase specifically loaded the tAR? amber suppressant tirosma mu it.an ^ te i (mutR? AACUA. >) with e-l ami.noá-ci.d, or not natura? l "?" 02- Phe. The mutant synthetase was derived from the selection of a mutant MjTyrRS library. The positions for mutagenesis in that mutant library were chosen in view of the analysis of the crystal structure of a MjTyrRS mu * t.an «t-e loading is ilec« t-i • v'amen-te mutR? ACUA with p-bromophenylalanine. After several rounds of positive selection and negative using CUA and the mutant MjTyrRS library in the presence or absence of 1 mM? 02-Phe, respectively, a clone was developed whose survival at a high concentration of chloramphenicol (90 μg / mL) was dependent on the presence of? 02-Phe. In addition, green fluorescence was only observed for the selected clone in the presence of? 02-Phe with a reporter T7 / GFPuv with an amber selector codon in place within the reporter gene. This result suggests that the synthetase developed has higher specificity for N02-Phe than for any other natural amino acid. Cloning of the clone revealed the following mutations in this evolved synthetase: Tyr32- Leu Glul07- Ser Aspl58- Pro Ilel59 ^ Leu Hisl60- > Asn Leul62-Glu The nucleotide sequence of this clone is provided in Table 5, SEQ ID NO: 11, and the corresponding amino acid sequence is provided in Table 5, SEQ ID NO: 10. To test the ability of the synthetase evolved (mutN02 -PheRS) and CUA to selectively incorporate N02-Phe to proteins, an amber retention codon was substituted at a permissive site (Lys7) in the gene the Z domain protein with a C-terminal exam His tag. The cells transed with mutN02-PheRS, CUA and the Z domain gene were cultured in the presence of 1 mM N02-Phe in minimal GMML medium. The mutant protein was purified using an affinity column of Nl2 + and subsequently analyzed by SDS-PAGE (see, figure 2) and MALDI-TOF (figure 3). The observed mass (m / e = 7958) of the MALDI-TOF analysis coincides with the expected mass (m / e = 7958) the Z domain protein incorporated with N02-Phe. No Z domain was obtained in the absence of N0 -Phe (see, figure 1), indicating a very high fidelity in the incorporation of the non-natural amino acid. Next, the feasibility of using N02-Phe incorporated as a fluorescence quencher was examined. Of the reported fluorophore counterparts of N02-Phe such as tyrosine, tryptophan, 1-pirenilalanine, and β-anthraniloyl-1-a, β-diaminopropionic acid, the tryptophan / N02 pair was chosen to incorporate a leucine zipper model GCN4, which s a parallel coiled coil homodimer. The DNA binding region of the GCN4 gene (676-840 bp), which does not encode any tryptophan, was cloned from the yeast genome into the pET-26b protein expression vector. Subsequently, site-directed mutagenesis was used to substitute amino acids in this protein at specific sites with either tryptophan or the unnatural amino acid N02-Phe (encoded by the TAG selector codon). The GCN4 expression vector also as a plasmid containing both mutN02-PheRS and CUA were co-transed to BL21 cells of E. coli (DE3), which were cultured in the presence of 1 mM N02-Phe in medium minimum of GMML. Cumulative GCN4pl mutant proteins were purified using a Ni2 + affinity column and confirmed by SDS-PAGE and MALDI-TOF analysis. The stable-state fluorescence spectra were measured the purified mutant proteins. Figure 4A shows the fluorescence spectrum of the 22Trp mutant protein alone and that of the mixture of 22Trp and 22N02-Phe mutants, while Figure 4B shows the fluorescence spectrum of the 55Trp mutant protein and the spectrum of the mutants 55Trp and 22N02-Phe. A different fluorescence switch off was observed in the 22Trp / 22N02-Phe mutant pair; On the other hand, no significant fluorescence quenching was obtained the pair of mutant 55Trp / 22N02-Phe. This result clearly shows that the fluorophore / quencher interaction between the pair of Trp / N02-Phe is distance dependent. Thus, this system can be easily applied to the study of protein folding and protein-protein as well as protein-ligand interactions.

EXAMPLE 3 Orthogonal translation components the in vivo incorporation of the active 3-amino-L-tyrosine redox amino acid to proteins in E. coli The present example describes compositions and methods the biosynthetic incorporation of 3-amino-L-tyrosine (see, figure 1, also written as NH2-YRS) to proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this unnatural amino acid were isolated that function in an E. coli host cell system. This unnatural amino acid side chain is easily oxidized to the corresponding semiquinone and quinone, so it can be used both probe and to manipulate the process of electron transfer in proteins. The oxidized quinone can be efficiently conjugated with acrylamide by means of a hetero-Diels-Alder reaction. This last property provides another use, that is, where the non-natural amino acid serves as a handle chemical modification of proteins. New orthogonal synthetases were derived from tyrosyl tyrosyl-tRNA synthetase from M. jannaschii and were used in conjunction with the suppressor tANNCuA of M. jannaschii (SEQ ID NO: 1). This new orthogonal pair has no affinity or has very low affinity any of the common amino acids (this is, which are presented in a stable way in nature). The derived orthogonal synthetase tRNA selectively charges the amber suppressor tARNCUA with 3-amino-L-tyrosine. The aminoacylated suppressor tRNA (that is, the charged rAR?) Is used as a substrate by the endogenous E. coli translation apparatus for incorporate 3-amino-L-tyrosine in response to the retention codon amber TABLET (a selector codon) found in a transcript. The orthogonality of these tRNA / synthetase pairs ensures that neither the tRNA nor the synthetases cross-react with endogenous E. coli tRNAs or synthetases and that the non-natural amino acid is only administered in response to TAG. The new synthetases were isolated using protocols that have been previously described. A library of tyrosyl-tRNA-synthetase mutants of M. jannaschii was generated by mutagenesis of the tyrosyl tRNA-synthetase of M. jannaschii was generated by mutagenesis of wild-type tyrosyl-tRNA-synthetase from M. jannaschii. The mutagenesis consisted of randomizing active site residues predicted based on the crystal structure of other aminoacyl-tRNA-synthetase molecules. Following the mutagenesis, the synthetase cluster in the mutant library was subjected to multiple rounds of positive and negative selection. This selection produced a synthetase clone that had the ability to load the O-tRNA with 3-amino-L-tyrosine. This selected synthetase clone has the amino acid sequence shown in Table 5, SEQ ID? O: 12 and has the polynucleotide sequence shown in Table 5, SEQ ID? O: 13.

EXAMPLE 4 Orthogonal translation components for the in vivo incorporation of the amino acid of p-carboxymethyl-L-phenylalanine which mimics phosphotyrosine to E. coli proteins The present example describes compositions and methods for the biosynthetic incorporation of p-carboxymethyl-carboxymethyl-L- phenylalanine (see Figure 1, also written as pCMF) to proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this unnatural amino acid were isolated that function in an E. coli host cell system. This side chain of non-natural amino acids can be used as a stable imitator for tyrosine phosphorylation. Tyrosine phosphorylation plays an important role in regulating cell signal transduction in a wide range of cellular processes, such as cell growth, metabolic regulation, transcriptional regulation and proliferation. Tyrosine phosphorylation is a reversible process in vivo. The tendency to deform tyrosine by endogenous tyrosine phosphatases interferes with studies of the effects of tyrosine phosphorylation, thus preventing the interpretation of these studies. The amino acid p-carboxymethyl-L-phenylalanine is a phosphotyrosine mimic, is permeable to the cell and also does not serve as a substrate for phosphatases of tyrosine. This non-natural amino acid, when incorporated into proteins, can be used to generate protein mutants that are constitutively active. This non-natural amino acid can also be used in the context of phage display or phage display to select inhibitors to the tyrosine phosphatase protein from peptide libraries containing p-carboxymethyl-L-phenylalanine. New orthogonal synthetases were derived from tyrosyl-tRNA synthetase from M. jannaschii and were used in conjunction with the suppressor tANNCuA of M. jannaschii. These new orthogonal pairs have no affinity or have very low affinity for any of the common amino acids (that is, they occur stably in nature). The derived orthogonal synthetases tARN selectively load the amber suppressor tARNCUA with p-carboxymethyl-L-phenylalanine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate p-carboxymethyl-L-phenylalanine in response to an amber TAG retention codon (a selector codon). ) found in a transcript. The orthogonality of these tRNA / synthetase pairs ensures that neither the tRNA nor the synthetases cross-react with endogenous E. coli tRNAs or synthetases and that the unnatural amino acid is only administered in response to TAG. A search for orthogonal synthetases was undertaken which have the ability to specifically load an orthogonal tRNA with p-carboxymethyl-L-phenylalanine. This search used protocols that have been previously described. A library of tyrosyl-tRNA-synthetase mutants of M. jannaschi i was generated by mutagenesis of the wild-type tyrosyl-tRNA-synthetase of M. jannaschii, where the mutagenesis consisted of randomizing active site residues predicted based on the crystalline structure of other aminoacyl-tRNA-synthetase molecules. Following mutagenesis, the mutant synthetase library was passed through multiple rounds of positive and negative selection. This selection produced five synthetase clones that have the ability to load the O-tRNA with p-carboxymethyl-L-phenylalanine. These synthetase clones were sequenced and the amino acid sequences were determined, as shown in Table 5. The amino acid sequences of these O-RS clones are provided in SEQ ID NOS: 14, 16, 18, 20 and 22. The nucleotide sequences of these same O-RS clones are provided in SEQ ID NOS: 15, 17, 19, 21 and 23.

EXAMPLE 5 Orthogonal translation components for the in vivo incorporation of the non-natural amino acid biphenylalanine hydrophobic to proteins in E. coli.

The present example describes compositions and methods for the biosynthetic incorporation of biphenylalanine (see, figure 1) into proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschi i to incorporate this non-natural amino acid were isolated that function in a host cell system of E. coli. The unnatural amino acid of biphenylalanine has a large aromatic side chain. Hydrophobic interactions are one of the main forces that drive the protein-protein-protein folding interactions (the other major forces are electrostatic interactions, hydrogen bonds and van der waals forces). Hydrophobic interactions are involved in many biological events, such as transport of proteins through cell membranes, protein aggregation and enzyme catalysis. The hydrophobicity of biphenylalanine is higher than any of the twenty common amino acids. The incorporation of biphenylalanine into proteins is a useful tool in the study and modulation of intramolecular and intermolecular hydrophobic packaging interactions in proteins. New orthogonal synthetases were derived from tyrosine-tRNA synthetase from M. jannaschii and are used in conjunction with the suppressor tRNANAT of M. jannaschii previously described. These new orthogonal pairs have no affinity or have very low affinity for any of the common amino acids (that is, they occur in a stable manner in nature). The derived orthogonal synthetases tARN selectively load the amber suppressor tARNCUA with biphenylalanine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA is used as a substrate for the translation apparatus of endogenous E. coli to incorporate biphenylalanine in response to the amber retention codon TAG (a selector codon) found in a transcript. of these tRNA / synthetase pairs ensures that neither the tRNA nor the synthetases cross-react with endogenous E. coli tRNAs or synthetases and that the unnatural amino acid is only supplied in response to TAG.A search was made for orthogonal synthetases that have the ability to specifically load an orthogonal tRNA with biphenylalanine This search used previously described protocols A library of tyrosyl-tRNA-synthetase mutants of M. jannaschii was generated by mutagenesis of the M-tyrosyltaRNA synthetase. jannaschii wild type, where the mutagenesis consisted of randomizing the active site residues predicted based on the crystalline structure of other aminoacyl tRNA-synthetase molecules. Following the mutagenesis, the mutant synthetase library was made up through multiple rounds of positive and negative selection. This selection produced seven synthetase clones that had the ability to load the O-tRNA with biphenylalanine. These synthetase clones were sequenced, as shown in Table 5. The amino acid sequences of these O-RS clones are given in SEQ ID NOS: 24, 26, 28, 30, 32, 34 and 36. The sequences of Corresponding nucleotides of these same O-RS clones are provided in SEQ ID NOS: 25, 27, 29, 31, 33, 35 and 37.

EXAMPLE 6 Orthogonal translation components for the in vivo incorporation of the unnatural metal chelating amino acid bipyridylalanine into proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of bipyridylalanine (see Figure 1) into proteins using the translation machinery of E. coli host cell. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this unnatural amino acid were isolated that function in an E. coli host cell system. The unnatural amino acid bipyridylalanine has the ability to chelate metal ions. The N, N-bidentate portion of this amino acid side chain is a strong chelator for transition metal ions, such as Cu2 +, Fe2 +, Ni2 +, Zn2 + and Ru2 +, etc. This amino acid metal chelator can be used to (1) introduce active or electrophilic redox metal ions to proteins, (2) form fluorescent metal ion complexes such as Ru (bpy) 3, or (3) moderate the metal ion-dependent dimerization of bipyridylalanine-containing proteins . New orthogonal synthetases were derived from tyrosyl tRNA synthetase from M. jannaschi i and were used in conjunction with the suppressor tANNCUA of M. jannaschii. The new orthogonal pairs have no affinity or have very low affinity for any of the common amino acids (that is, they occur stably in nature). The derived orthogonal synthetase tARN selectively charges the amber suppressor tARNCUA with bipyridylalanine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate bipyridylalanine in response to the amber TAG retention codon (a selector codon) found in a transcript. The orthogonality of these pairs of tAR? / Synthetases ensures that neither the TAR? nor do the synthetases react in a cross-way with the tAR? of endogenous E. coli or synthetases and that the non-natural amino acid is only administered in response to TAG. We undertook an orthogonal synthesizer search that has the ability to specifically load a TAR? orthogonal with bipyridylalanine. This search used protocols that have been previously described. A library of tyrosyl tRNA-synthetase mutants of M. jannaschii was generated by mutagenesis of wild-type tyrosyl-tAR? -syntatase of wild-type M. jannaschii, where the mutagenesis consisted of randomizing predicted active site residues based on the crystalline structure of other molecules of aminoacyl-tAR? - synthetase. Following mutagenesis, the mutant synthetase library was passed through multiple rounds of positive and negative selection. This selection produced two synthetase clones that had the ability to load the O-tAR? with bipyridylanine. These synthetase clones were sequenced, as shown in Table 5. The amino acid sequences of these O-RS clones are provided in SEQ ID NOS: 38 and 40. The corresponding nucleotide sequences of these same O-RS clones. are provided in SEQ ID NOS: 39 and 41.

EXAMPLE 7 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid fluorescent 1,5-dansilalanine to proteins in yeast host cells. The present example describes compositions and methods for the biosynthetic incorporation of 1,5-dansilalanine (see, figure 1) into proteins using the yeast host cell translation machinery. New pairs of synthetase / tRNA Orthogonal derivatives of E. coli to incorporate this unnatural amino acid were isolated that function in the yeast host cell system. Fluorescence has become one of the most important detection signals in biotechnology due to its high sensitivity and safety management. In addition, processes such as fluorescence resonance energy transfer (FRET) or fluorescence polarization enable real-time analysis of biomolecular binding events, movements or conformational changes. The current fluorescent methodology for studying proteins in vivo frequently depends on fusion constructs with large fluorescent proteins. Alternatively, small organic labels can be used to minimize structural disturbance, but exhibit poor regioselectivity, are cytotoxic and require the introduction of portions of dye binding protein and are rather restricted to the protein surface. In contrast, a fluorescent amino acid does not necessarily contain groups with cytotoxic potential, its introduction is only a minor alteration of the protein structure and specific labeling is possible at any position of the protein in vivo. The present invention provides orthogonal translation system components that incorporate the fluorescent amino acid 1,5-dansil-modified (see, Figure 5A) in increasing polypeptide chains in yeast. This non-natural amino acid can also be identified by its IUPAC nomenclature: 2-ammo-3- (5-d? Met? Lam? No-naphthalen-1-sulfon? Lam?) -propionic acid. The dansyl chromophore has interesting spectral properties, which include an exceptionally high excitation and emission maxima (> 200 nm) and a high emission intensity dependence on the polarity of the environment. This makes it suitable for studying changes protein conformations or binding events where the local protein environment and thus the polarity is affected The synthesis of the non-natural amino acid was obtained in a two-step procedure that includes the coupling of N-Boc-ammoalanma to dansilchloride using triethylamine in dichloromethane and subsequent acid deprotection with TFA in dichloromethane. New smtatases to incorporate 1,5-dans? lalan were isolated using previously described protocols, see for example, Wu et al, Journal of the American Chemical Society 126: 14306-14307 (2004), and International application No. PCT / US 2005/034002, filed on September 21, 2005 by Deiters et al. A mutant E. coli leucyl-tARN smtetase clone (clone B8) which showed initial loading activity was isolated from a randomized E. coll leucyl-tRNA library of E. coll in a yeast host cell system. See, Table 5 and SEQ ID NOS: 42 and 43. The sites in the leucyl-tRNA synthetase library of E. coli mutant were M40, L41, Y499, Y527 and H537. Additional mutations (caused during library construction) found in all clones through the library were H67R, N196T, R262A and S497C. However, the B8 mutant E. coli synthetase exhibited background activity towards one or more natural amino acids with a leucine-like weight as judged by MALDI TOF MS of expressed human superoxide dismutase of expressed protein bearing a permissive amber codon. in position 33 (hSOD-33TAG-His6). Theoretical docking studies with dansilalanine-AMP amide and a crystal structure of the homologous leucyl-tRNA synthetase from Thermus thermophilus (T. th.) Suggested the formation of an extended binding cavity that binds the ligand through primarily hydrophobic interactions without involvement of p-stacked to the naphthyl moiety (see, Figure 5B). A reading-proof activity is present in the E. coli leucyl-tRNA synthetase and since activation and loading activity towards 1,5-dansilalanine was already developed in the selected mutant, a strategy aimed at selective removal was devised of activated or charged natural amino acids when remodeling the editing site. It was contemplated that the observed background was due to the incorporation of leucine as suggested by MALDI TOF MS. The crystal structures of the leucyl-tRNA synthetase homologue of T. th. and mutational studies suggest that a simple steric block of nonpolar amino acids to the β -methyl side chain prevents activated or charged leucine from binding to the hydrolytic site (Lmcecum et al., Mol Cell., 4: 951-963 [2003] ]). To increase the hydrolytic activity towards leucma, residues T252 and V338 in the smtetase editing domain of E. coll were exchanged to alamna by Quikchange mutagenesis in order to expand the binding cavity (see, Figure 6A). The V338A smtetase (see, Table 5, SEQ ID NOS: 46 and 47) showed no significant difference in expression studies using the human protein superoxide dismutase model (hSOD), while the T252A smtetase (see, Table 5, SEQ ID NOS: 44 and 45) showed a marked reduction in the background (see figure 6B). The high selectivity of this mutant was further confirmed by MALDI TOF MS from hSOD-33TAG-H? S6. Thus, the invention provides novel mutant tRNA-smtetases derived from E. coll leucyl-tARN smtetase which have the ability to biostatically incorporate 1,5-dansilalanine to proteins using the yeast host cell translation machinery (e.g., Saccharomyces cerevisiae). ).

EXAMPLE 8 Orthogonal translation components for the in vivo incorporation of the non-natural photoenzylated amino acid o-nitrobenzyl serine to proteins in yeast host cells The present example describes compositions and methods for the biosynthetic incorporation of o-nitrobenzyl serine (see, figure 1) to proteins using the yeast host cell translation machinery. New orthogonal synthetase / tRNA pairs derived from E. coli to incorporate this unnatural amino acid were isolated that function in the yeast host cell system. The investigation of the function of a specific gene in living organisms depends mostly on its deactivation or activation and the study of the resulting effects. Classical gene expulsion studies point the gene to the DNA level, leading to the deactivation of the production of all the encoded protein variants and do not allow real-time investigation of the resulting effects. In recent years, the use of small organic molecules has dramatically increased the specificity of genetic deactivation. Using such tools, a single variant of protein (or a single domain of that variant) can be targeted and the effects can be investigated in real time after the addition of the molecule. The introduction of photoengineered amino acids to Proteins such as transient activable expulsions can further increase the accuracy of such studies. Using chemical expulsion strategies, the diffusion time of the compound to its target protein can be speed limiting and it is only possible to investigate an entire cell. In contrast, the photodetection of specific amino acids can be performed on a rapid time scale and specific compartments of a cell can be investigated using pulsed and highly focused laser light. A mutant E. coli leucyl-tRNA synthetase has been previously developed from a mutant E. coli leucyl-tRNA synthetase library in yeast host cells that specifically recognizes the caged cysteine derivative o-nitrobenzylcysteine, also written as o-NBC (See, Wu et al., Journal of the American Chemical Society 126: 14306-14307 (2004); and international application No. PCT / US2005 / 034002, filed on September 21, 2005, by Deiters et al. ).

To expand the application of this procedure, the evolution of an aminoacyl-tRNA synthetase that specifically incorporates o-nitrobenzyl-serine (or NBS) was contemplated. When incorporated genetically into proteins, this non-natural photoen.julled amino acid could be used to photoregulate any function involving serine residues, for example but not limited to, serine phosphorylation by kinases, which they represent one of the most important chemical markers in signal transduction routes. The amino acid oNBS can be synthesized by coupling o-nitrobenzyl bromide to Boc-N-Ser-O-tBu in DMF using NaH as a base and subsequent acid deprotection with TFA in methylene chloride ba or the presence of tretilsilane as a scrubber with 52% Overall performance The mutant E. coll leucyl-tRNA smtetase evolved for incorporation of oNBC (clone 3H11, see Table 5, SEQ ID NOS 48 and 49) already exhibited some limited activity to incorporate oNBS, but with approximately twice reduced efficiency in comparison with an amino acid of oNBC To evolve a more efficient translation system of oNBS, the selected 3H11 clone smtetase was diversified by error-prone PCR, again using three different mutations, to induce one, two or five mutations per gene, producing a global diversity of 1 x 107 clones. The positions in the leucyl-tRNA-smtetase enzyme that were targeted for randomization were M40, L41, Y499, Y527 and H537. The protocols used herein follow the general methodologies described in the art, for example Wu et al., Journal of the American Chemical Society 126: 14306-14307 (2004); and international application No. PCT / US2005 / 034002, filed on September 21, 2005, by Deiters et al.

The selection of the new mutant synthetase library produced an improved synthetase (clone G2-6) with enhanced efficiency of oNBS incorporation twice. Sequencing of clone G2-6 identified five additional mutations in the whole enzyme compared to the initial 3H11 synthetase starting material (positions S31G, T247A, T248S, M617I and V673A). Additional mutations (caused during the construction of the library) found in all clones throughout the library were also observed as follows: H67R, N196T, R262A and S497C. The complete amino acid and nucleotide sequences of this synthetase isolate are provided in Table 5, SEQ ID NOs: 50 and 51. This improved mutant synthetase is illustrated schematically in Figure 7A, and the improvement in oNBS incorporation activity in the G2-6 mutant synthetase is illustrated experimentally in Figure 7B. The selective incorporation of oNBS was further confirmed by MALDI MS again using hSOD as a model system for expression studies. Thus, the invention provides a novel mutant tRNA-synthetase derived from E. coli leucyl-tRNA synthetase which has the ability to biosynthetically incorporate or NBS to proteins using the yeast host cell translation machinery (eg, Saccharomyces cerevisiae).

EXAMPLE 9 Orthogonal translation components for the in vivo incorporation of photoenjalated non-natural amino acid 0- (2-nitrobenzyl) -L-tyrosine to proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of 0- (2 - nitrobenzyl) -L-tyrosine (see, figure 1) to proteins using the Archae synthetase species and the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschi i to incorporate this non-natural amino acid were isolated that function in the host cell system of E. coli. "Caged proteins" are modified proteins whose biological activity can be controlled by light, usually by photolytic conversion from an inactive form to an active form. This is particularly useful since irradiation can be easily controlled in timing, location and amplitude, allowing detailed studies of protein function (for reviews, see Shigeri et al., Pharmacol. Therapeut., 2001, 91:85; Curley and Lawrence. Pharmacol, Therapeut, 1999, 82: 347, Curley and Lawrence, Curr. Op. Chem. Bio, 1999, 3:84, "Caged Compounds," Methods in Enzymology, Marriott, G., Ed., Academic Press: New York , 1998, V. 291, and Adams and Tsien, Annu, Rev. Physiol., 1993, 55: 755).

The most common caging groups are 2-nitrobenzyl groups (see, Bochet, J. Chem. Soc., Perkin 1 2002, 125; Givens et al., Methods in Enzymology 1998, 291, 1; and Pillai, Synthesis 1980, 1). , which are installed in hydroxy, carboxy, thio, or amino groups of polypeptides or proteins and are easily cleaved in the irradiation with non-photodating UV light. Previously, the caged proteins were produced by chemical modification of isolated proteins without position control on the set-up of the pool and also the majority resulting in the incorporation of multiple set-up groups (for example Self and Thompson, Nature Med. 1996, 2, 817). Other examples employ in vitro incorporation of a caged amino acid using a nonsense codon excision technique (see, Philipson et al., Am. J. Physiol., Cell. Physiol., 2001, 281, C195; Pollitt and Schultz Angew. Int Ed. 1998, 37, 2105; Cook et al., Angew, Chem. Int. Ed. 1995, 34, 1629). Since the aminoacylated tRNA has to be synthesized chemically, only small amounts of protein are accessible and in vivo studies are limited. The use of orthogonal translation system technology has overcome the limitations inherent in these technologies. Using cellular systems, non-natural amino acids can be specifically incorporated into the site with high fidelity protein translation in vivo through addition of new components to the translation machinery of E. coli (for review, see, for example, Wang and Schultz, Angew, Chem. Int. Ed. 2004, 44, 34; Cropp and Schultz, Trend. Gen. 2004, 20, 625; and Wang and Schultz, Chem. Commun. 2002, 1). The present example describes the additional of a photoenzylated tyrosine, O- (2-nitrobenzyl) -L-tyrosine (see Figure 1), to the genetic code of E. coli. Tyrosine is an important amino acid in tyrosine kinase and phosphatase protein substrates, is a residue in essentially several active enzyme sites and is frequently located at protein-protein interfaces. The irradiation of O- (2-nitrobenzyl) -L-tyrosine (synthesized from l-tyrosine as described in Miller et al., Neuron 1998, 20, 619) at 365 nm induces excision of the benzylic CO bond and rapid formation of the de-hindered amino acid (tM = 4 min, see support information), as schematically illustrated in Figure 8. The photodetention of O- ( 2-nitrobenzyl) -L-tyrosine can be observed experimentally, as illustrated in the experimental results shown in figure 9. As shown in this figure, the photodensation of O- (2-nitrobenzyl) -L-tyrosine was studied by irradiation of a 0.2 mM solution in water (a cavity of a six cavity box) using a portable UV lamp (365 nm at 10 mm distance). Aliquots were taken at points in time specific and analyzed by LC / MS. The concentrations of O- (2-nitrobenzyl) -L-tyrosine (squares) and de-entrained species (circles) are shown in the figure. 50% unraveling is obtained after approximately four minutes. The tyrosyl-tRNA-synthetase from Methanococcus jannaschii (MjYRS) was used as a starting point for the generation of an orthogonal synthetase that accepts O- (2-nitrobenzyl) -L-tyrosine, but not any of the twenty common amino acids as a substrate. MjYRS does not aminoacylate any endogenous E. coli tRNA with tyrosine, but aminoacylates a mutant tyrosine amber suppressor (mutRNACuA) • To alter the specificity of MjYRS to selectively recognize O- (2-nitrobenzyl) -L-tyrosine, a library of approximately 109 YRS mutants was generated by randomizing six residues (Tyr32, Leu65, Phel08, Glnl09, Aspl58 and Leul62) into the tyrosine binding cavity, based on the crystal structure of the YRS / tRNATyr-tyrosine complex of M. jannaschii (Zhang et al., Prot. Sci. 2005, 14, 1340; Kobayashi et al., Nat. Struct. Biol. 2003, 10, 425). These six residues were chosen for their close proximity to the para position of the tyrosine phenyl ring, among which Tyr32 and Aspl58 form hydrogen bonds with the hydroxyl group of tyrosine. It is expected that mutations of these residues expand the substrate binding cavity of the synthetase to specifically recognize O- (2-nitrobenzyl) -L- tyrosine and other non-natural amino acids. Active synthetase variants were selected from the mutant MjYRS library using chloramphenicol acetyl transferase (CAT) and barnase reporter systems for positive and negative selections, respectively. After five rounds of positive and negative selection alternating 96 clones were selected on when to a phenotype in the presence and absence of O- (2-nitrobenzyl) -L-tyrosine. Three synthetases were further characterized using an in vivo analysis based on the suppression of the Aspll2TAG codon in the CAT gene. Expression of E. coli from the three pairs of MjYRS / mutRNACUA survived in chloramphenicol with IC50 values of 110 mg / L and less than 10 mg / L in the presence and absence of O- (2-nitrobenzyl) -L-tyrosine ( 1 mM), respectively. The large difference in chloramphenicol resistance suggests a substantially in vivo specificity of the selected synthetase / tRNA pairs for O- (2-nitrobenzyl) -L-tyrosine insertion with respect to all twenty natural amino acids in response to an amber codon. The nucleic acids encoding these three O- (2-nitrobenzyl) -L-tyrosine-tRNA synthetases were sequenced and their amino acid sequences were deduced. The complete amino acid sequences of the three ONBY synthetase clones are provided in Table 5, SEQ ID NOs: 52-54. The results of this sequence are shown in Table 3.

TABLE 3 Conceivably, the Tyr32-> mutations Gly32 / Ala32 and Aspl58- > Glul58, Alal58, or Serl58 result in the loss of hydrogen bonds between Tyr32, Aspl58 and the natural substrate tyrosma, thus disfavoring their binding. To measure the fidelity and efficiency of the three ONB-MjYRS, O- (2 -trobenzyl) -L-tyrosma was incorporated in response to an amber codon in position four in a mutant sperm whale myoglobin gene hexahistidma marked C-thermically . To express recombinant protein, the plasmid pBAD / JYAMB-4TAG (which encodes the mutant sperm whale myoglobin gene with an arabmosa promoter and an rrnB terminator, the tyrosyl-tRNANcuA on an Ipp promoter and a rrnC terminator, and a tetracyclic-resistant marker) was co-transfected with a pBK vector (which encodes the mutant synthase and a kanamycin-resistant gene) in E. coli DH10B E. coli in the presence of both the synthetase pair / mutRNACuA and 0- (2-nitrobenzyl) -L-tyrosine (1 mM). The cells were amplified in a Luria-Bertani medium (5 mL) supplemented with tetracycline (25 mg / L) and kanamycin (30 mg / L), washed with phosphate buffer and used to inoculate 500 mL of minimal medium of liquid glycerol (GMML; minimum glycerol medium supplemented with 0.3 mM leucine) containing the appropriate antibiotics, photoenjalated tyrosine (1 mM), and arabinose (0.002%). The cells were cultured to saturation and then harvested by centrifugation. The purified mutant myoglobin protein was obtained by Ni-NTA affinity chromatography at a yield of about 2-3 mg / L and judged to be > 90% homogeneous by SDS-PAGE and Gelcode blue staining. The yield is comparable to the expression of myoglobin using the Mj YRS / wild type mutRNACUA pair that suppresses the same amber codon. Myoglobin was not detectable if the non-natural amino acid was retained or in the presence of 1 mM tyrosine, revealing a very high selectivity of all three synthetases by 0- (2-nitrobenzyl) -L-tyrosine (see, figure 10). To further confirm the identity of the photoenjalated protein specifically at the site, a different myoglobin mutant with an amber codon in Gly74 (due to the properties of higher mass spectrometry) was expressed in the presence of pONB-MjYRS-1, tRNACuA, and 0- (2-nitrobenzyl) -L-tyrosine (1 mM). The 74TAG myoglobin mutant was expressed, under the same conditions as the 4TAG mutant, using the pONB-1 synthetase in the presence of 0- (2-nitrobenzyl) -L-tyrosine (1 mM) and purified by nickel affinity column. Protein bands were visualized by dyeing Gelcode blue from an SDS-PAGE gel and excised from the polyacrylamide gel. The gel pieces were cut into 1.5 mm cubes and subjected to hydrolysis of trypsin, essentially as described (Shevchenko et al., Anal.Chem. 1996, 68, 850-858). Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis performed on a Finnigan LCQ Deca ion trap mass spectrometer (Thermo Finnigan) equipped with a Nanospray HPLC (Agilent 1100 Series) . The precursor ions corresponding to the individual and double charged ions of the peptide HGVTVLTALGJILK containing the non-natural amino acid (denoted as J) were separated and fragmented with an ion trap mass spectrometer. The LC-MS / MS analysis shows tyrosine in position 74 (tryptic peptide HGVTVLTALGYILK). The ionic masses of the fragment could be assigned, indicating the specific incorporation at the site of tyrosine (3) in position 74 (see Figures HA and 11B). The detection of Tyr74 is more likely due to a fragmentation of labile benzyl ether in O- (2-n? trobenzyl) -L-tyrosine during MS analysis. To confirm the previous incorporation of the caged amino acid 0- (2-methylbenzyl) -L-tyrosma, the deuterated derivative was synthesized and used in an expression of the same myoglobulin mutant under identical conditions. Then the protein was trypsinized and subjected to an analysis by mass spectrometry. An allocation of the fragment ion masses revealed the incorporation of d2-t-rosma in position 74 of myoglobulin, unambiguously demonstrating the incorporation of unnatural amino acid 2 (see Figures 12A and 12B). The LC MS / MS analysis did not indicate incorporation of any natural amino acid in this position, providing additional evidence for the high fidelity of the evolved smtetase. Additionally, the photochemical activation of a m-protein that has 0- (2-mtrobenzyl) -L-tyrosma incorporated can be demonstrated by using lacZ as a reporter gene, β-galactosidase from E coll exhibits an essential t-ray at position 503 (Juers et al., Biochemistry 2001, 40, 14781; Penner et al., Biochem Cell Biol. 1999, 77, 229). The corresponding codon was mutated to an amber retention codon TAG for the incorporation of the caged 0- (2-nitrobenzyl) -L-tyrosma. The β-galactosidase is verified before and after tyrosine desenj aulamiento. The β-galactosidase activity is restored immediately after irradiation in vivo.

EXAMPLE 10 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid p-cyanophenylalanine to proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of p-cyanofemilalamine (see, figure 1, also written as 4-c? anofe lalanma) to proteins using the host cell translation machinery of E coli New orthogonal smtetase / tRNAs derived from M. j annaschu to incorporate this non-natural amino acid were isolated that work in a host cell system of E. coli. The cyano group is an excellent local environmental IR probe, since its CN stretch vibration (v2) undergoes a frequency shift of the order of ten wave numbers when it is moved from the hydrophobic to hydrophilic surroundings Getahun et al., "Usmg Nitrile-Derivatized Ammo Acids as Infrared Probes of Local Environment ", JACS 125, 405-411

[2003]) Para-cyanophenalamine (position 4) and meta-cyanophelanoma (position 3) cyanofemulamine are thus useful for studying an assortment of protein properties in which protein-protein binding, protein conformation and folding are included or hydrophobic crushing. The forms for and goal of cyanophenylalanma can exist in both polar and hydrophobic environments, while that in a chain of peptide and its effects on the conformation are negligible. Thus, it is already likely that they reside in the same environment as the wild-type residue that they replace in a protein or peptide. In addition, the CN stretch vibration of the components is narrow, does not overlap with any other protein absorptions, is extensively decoupled from the other protein vibrations and is quite sensitive to changes in solvent polarity. For these reasons, both are excellent tools for these conformational peptides.

Aromatic Nitriles as Local-IR IR Probes in Small Peptides Getahun et al. (Getahun et al., "Using Nitrile-Derivatized Amino Acids as Infrared Probes of Local Environment ", JACS 125, 405-411 [2003]) have shown (see, figure 13) that cyano-phenylalanine cyan stretching vibration is ten higher wave numbers in water than in THF. Mastoparan residues x (MPx) is mutated to incorporate para-cyanophenylalanine to its lipid binding portion, the cyano stretch is at 2229.6 cm "1 when MPx is linked to a lipid bilayer of POPC. In water, the CN stretch vibration of the MPx PheCN mutant occurs at 2235.7 cm "1. In short, the cyano stretch in a hydrated peptide is similar to the cyano stretch of free para-cyanophenylalanine in water, whereas the cyano stretch of PheCN in a buried peptide is similar to the cyclic stretch THF of free PheCN (Tucker et al., "A New METHOD for Determining the Local Environment and Orientation Of Individual Side Chains of Membrane-Binding Peptides ", JACS 126 5078-5079 [2004]). A new orthogonal synthetase was derived from tyrosyl tRNA synthetase from M. jannaschi i and used in conjunction with the suppressor tARNCUA from M. jannaschii previously described. This new orthogonal pair has no affinity or has very low affinity for any of the common amino acids (that is, they occur stably in nature). The derived orthogonal synthetase tRNA selectively charges the amber suppressor tARNCUA with p-cyanophenylalanine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate p-cyanophenylalanine in response to the amber TAG retention codon (a selector codon) found in a transcript . The orthogonality of these tRNA / synthetase pairs ensures that neither the tRNA nor the synthetases cross-react with endogenous E. coli tRNAs or synthetases and that the unnatural amino acid is only administered in response to TAG. We undertook a search for orthogonal synthetases that have the ability to specifically load a tRNA orthogonal with p-cyanophenylalanine. This search used protocols that have been previously described. A library of tyrosyl-tRNA-synthetase mutants of M. jannaschii was generated by mutagenesis of wild-type tyrosyl-tRNA-synthetase from M. jannaschi, where the mutagenesis consisted of randomizing active site residues predicted based on the crystalline structure of other aminoacyl-tRNA-synthetase molecules. Following the mutagenesis the mutant synthetase library was passed through multiple rounds of positive and negative selection. This solution produced a synthetase clone that had the ability to load the O-tRNA with p-cyanophenylalanine. This synthetase clone was sequenced and the amino acid sequence was determined, as shown in Table 5, SEQ ID NOs: 55 and 56). This mutant synthase shows the following substitutions in relation to the wild type synthetase sequence: Tyr32Leu, Leu65Val, Phel08Trp, Glnl09Met, Aspl58Gly and Ilel59Ala.

EXAMPLE 11 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid m-cyanophenylalanine into proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of m-cyanophenylalanine (see, Figure 1, also written as 3-c? anophenylalanine) to proteins using the host cell translation machinery of E. coli. New pairs of orthogonal smtetasa / tRNAs derived from M. j annaschu to incorporate this unnatural amino acid were isolated that function in a host cell system of E. coll. The cyano group is an excellent local environment IR probe, since its NC stretch vibration (v2) undergoes a frequency shift of the order of ten wave numbers when it is moved from hydrophobic to hydrophilic surroundings (Getahun et al, " Using Nitrile-Depvatized Ammo Acids as Infrared Probes of Local Environment ", JACS 125, 405-411 [2003]). For cyanofemulamine (position 4) and meta-cyanofemulalan (position 3), they are useful in the study of an assortment of protein properties, including protein-protein binding, protein conformation, and hydrophobic folding or crushing. The para and meta forms of cyanophenylalanine can exist in polar and hydrophobic environments insofar as they are in a peptide chain and their effects on conformation are negligible. Thus, it is likely that either one resides in the same environment as the wild-type residue that it replaces in a protein or peptide. In addition, the CN stretch vibration of the compounds is narrow, does not overlap with any other protein absorptions, is Widely decoupled from the other vibrations of the protein and is quite sensitive to changes in solvent polarity. For these reasons, both are excellent tools for peptide conformational studies.

AROMATIC NITRILES IN PROTEINS Following established established evolution protocols, a new pair of Methanococcus jannaschi i tRNATyrCuA ~ irosyl-RNA synthetase (TyrRS was developed that specifically incorporated into the meta-cyanophenylalanine site with high fidelity in response to an amber TAG codon. orthogonal pair has no affinity or has very low affinity for any of the common amino acids (that is, they occur stably in nature.) The derived orthogonal tRNA synthetase selectively charges the amber suppressor tARNCUA with m-cyanophenylalanine. aminoacylated (ie, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate m-cyanophenylalanine in response to the amber retention codon of TAG (a selector codon) found in a transcript. The orthogonality of these tRNA / synthetase pairs ensures that neither the tRNA nor the synthetases react in a cru The TAN of endogenous E. coli or synthetases and that the unnatural amino acid is administered only in response to a codon without amber sense, TAG.

The construction of the orthogonal smtetase has the ability to specifically load an orthogonal tRNA with m-cyanofemulala using protocols that have been previously described. A library of tyrosyl-tRNA-smtetase mutants of M. j annaschu was generated by tyrosyl-mutagenesis. RNA-smtetase from wild-type M. jannaschu, where the mutagenesis consisted of randomizing active site residues predicted based on the crystal structure of other ammoacyl-RNA-smtetase molecules. Following the mutagenesis, the mutant smtetase library was passed through multiple rounds of positive and negative selection. This selection produced a smtetase clone that had the ability to load the O-tRNA with m-cyanofemulase. This smtetase clone was sequenced and the amino acid sequence was determined (see, Table 5, SEQ ID NOs 57 and 58) This mutant smtetase follows the following substitutions in relation to the wild type smtetase sequence Tyr32H? S, H? S70Ser, Aspl58Ser, Ilel59Ser and Leul62Pro. An attempt was made to suppress a mutant Tyr7 - > TAG of the Z domain protein c terminal H? S6-et? Quetada both in the presence and in the absence of m-cyanophenylalanine and p-cyanofemulala, using their respective para tRNA / orthogonal smtetase. In both cases, full-length protein was produced in the presence of an unnatural amino acid, in so much that no product was detectable by staining Coomassie blue on an SDS-PAGE gel in the absence of the respective non-natural amino acid. In addition, IR spectra of this protein were obtained with both meta and para-cyanofemlalamine incorporated in position 7. After the background subtraction of the wild type Z-domain IR spectrum, the spectra shown in figures 14A and 14B are obtained. Figure 14A shows that the para-cyanophenylalanine has a single absorbance between the ends shown in Figure 13, suting that residue number seven falls along the surface of the protein, but does not point directly at the solution. Figure 14B shows the spectrum of meta-cyanofemulalanma as the sum of two Gaussian distributions with peaks at 2236 and 2228 cm "1. The value of R2 for the curves is greater than 0.99, an excellent curve fit and the figure data 14B thus sut that the meta-cyanofe lalamna has two confirmations.As evidenced by the peak at 2228 cm "1, a conformation places the cyano group in a hydrophobic region of the protein. The peak at 2236 cm 1 suts that the other conformation sites are in a hydrated environment.

EXAMPLE 12 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid p- (2-amino-1-hydroxyethyl) -L-phenylalanine to proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of p-1. (2-amino-1-hydroxyethyl) -L-phenylalanine (see, figure 1) to proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschi i to incorporate this non-natural amino acid were isolated that function in a host cell system of E. coli. The site-specific modification of proteins with biophysical probes, cytotoxic agents, cross-linking agents, and other agents that have been widely used to analyze the structure and function of protein and in the development of diagnostics, therapeutic agents, and high-throughput screening. A method for the selective modification of proteins involves the oxidation of an N-terminal serine or threonine to the corresponding aldehyde and subsequent coupling with hydrazine, alkoxyamine or hydrazide derivatives. Unfortunately, this method is limited since it can only be used to modify the N-terminal position of a protein. The procedure to place the critical aminoalcohol functional group of 2-amino-1- Hydroxyethyl on a side chain of the target protein will remove the limitation of selective protein modification on the N-terminus only with the additional benefit of controlling the position of the aminoalcohol group in the protein. A new orthogonal synthetase was derived from tyrosyl-tRNA synthetase from M. jannaschii and is used in conjunction with the suppressor tRNANUA of M. jannaschii previously described. This new orthogonal pair has no affinity or has very low affinity for any of the common amino acids (that is, they occur stably in nature). The derived orthogonal synthetase tRNA selectively charges the amber suppressor tARNcuA with p- (2-amino-1-hydroxyethyl) -L-phenylalanine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate p- (2-amino-1-hydroxyethyl) -L-phenylalanine in response to a codon amber retention TAG (a selector codon) found in a transcript. The orthogonality of this tRNA / synthetase pair ensures that neither the tRNA nor the synthetase cross-reacts with the endogenous E. coli RNA or synthetases and that the unnatural amino acid is incorporated only in response to an amber non-sense codon, TAG . We undertook a search for orthogonal synthetases that have the ability to specifically load an orthogonal tRNA with p- (2-amino-1-hydroxyethyl) -L-phenylalanine. This search used protocols that have been previously described. A library of tyrosyl-tRNA-synthetase mutants of M. jannaschii was generated by mutagenesis of wild type tyrosyl-tRNA-synthetase from M. jannaschii, where the mutagenesis consisted of randomizing predicted active site residues based on the structure crystalline of other molecules of aminoacyl-tRNA-synthetase. Following mutagenesis, the mutant synthetase library was passed through multiple rounds of positive and negative selection. This selection produced a synthetase clone that had the ability to load the O-tRNA with p- (2-amino-1-hydroxyethyl) -L-phenylalanine. That synthetase clone was sequenced and the amino acid sequence was determined (see, Table 5, SEQ ID NO: 59). This mutant synthase shows the following substitutions in relation to the wild type M. jannaschii synthetase sequence: EXAMPLE 13 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid p-ethylthiocarbonyl-L-phenylalanine into proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of p-ethylthiocarbonyl-L-phenylalanine (see, Figure 1) to proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this unnatural amino acid were isolated that function in an E. coli host cell system. A useful method for the generation of semisynthetic proteins is the natural chemical ligation in which two fully deprotected peptide fragments can be coupled by an amide bond under moderate physiological conditions at room temperature (Nilsson et al., Annu, Rev. Biophys. Biomol., Struct. 2005, 34, 91-118; Dawson et al., Science 1994, 266, 776-779). A variation of this method, called expressed protein ligation, in which one or both of the reaction partners have been produced by recombinant means, is useful for the synthesis of proteins consisting of more than 100 residues (Muir, Annu Rev. Biochem 2003, 72, 249-289; David et al., Eur. J. Biochem. 2004, 271, 663-677). In practice, both techniques require the presence of a C-terminal thioester, limiting these methods to modification in the C term of a peptide fragment. The placement of a reactive thioester group in any residue in a bacterially expressed peptide / protein would expand significantly in scope of these techniques, allowing for example the synthesis of cyclic or branched structures or the selective modification of side chains with biophysical probes, polyethylene glycols or various labels. Methods for the generation of proteins having thioester-containing side chains find use in that the thioester-containing side chains may be particular in subsequent chemical ligation reactions m vitro and possibly m vivo (Camarero and Muir, J. Am. Chem. Soc. 1999, 121, 5597-5598; Camarero et al, Bioorg Med Chem 2001, 9, 2479-2484; Scott et al., Proc. Nati Acad. Sci. USA 1999, 96, 13638-13643, Evans et al, J. Biol Chem. 2000, 275, 9091-9094, Yeo et al., Chem. Commun. 2003, 2870-2871).

SYNTHESIS OF P-ETILTIOCARBONIL-L-PHENYLALANIN The p-ethylthiocarboml-L-phenylalanm (structure 1, also called 4 - (ethylthiocarbonyl) -L-femlalamine) was synthesized in four stages (see, figure 15) starting from a-bromine acid -p-toluic commercially available (la) and N- (diflemlmethylene) glycine tert-butyl ester (le). These stages are summarized below.

Synthesis of S-ethyl 4- (bromomethyl) benzothioate (structure lb): To a solution of (2.15 g, 10.0 mmol) in THF (50 ml) is added thionyl chloride (2 ml, 28 mmol), followed by the addition of DMF (50 μl) and the reaction mixture was stirred for 5 hours at room temperature. The organic solvents were removed under reduced pressure until a white solid appeared, which was then dissolved in THF (50 ml) and the solution was cooled to 0 ° C. A solution of etantiol (0.78 ml, 10.0 mmol) and triethylamine (2 ml, 14 mmol) in THF (10 ml) was added dropwise in the course of minutes. The reaction mixture was stirred for another four hours and solvent was removed. Water (100 ml) and ether are added (200 ml). The organic phase was washed with H20 (2 x 50 mL), dried over NaSO4, and stirred under reduced pressure. The crude product was purified by flash chromatography on silica gel (8% ethyl acetate in hexane), yielding lb (2.18 g, 78%) as a colorless oil. NMR (400 MHz, CDCl3) d 7.94 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 4.50 (s, 2H), 3.07 (q, J = 7.6, 15.2 Hz, 2H), 1.35 (t, J = 9.2 Hz, 3H). Exact mass m / z calculated for C10H?: LBrOS 258.0 / 260.0, found (LC / MS) 259.1 / 260.1.

Synthesis of tert-butyl 2- (diphenylmethyleneamino) -3 - (4- (ethylthiocarbonyl) phenyl) propanoate (structure ld): a solution containing lb (0.455 g, 1.76 mmole), (0.47 g, 1.60 mmol), 18-crown-6 (0.42 g, 1.59 mmol) and anhydrous K2C03 (0.344 g, 2.50 mmol) in anhydrous CH3CN (10 mL) was stirred for 24 hours at room temperature. The organic solvents were removed under reduced pressure. Water (100 ml) and CH2C12 (200 ml) are added. The organic phase was washed with H20 (2 x 50 mL), dried over NaSO4, and stirred under reduced pressure. The crude product was used directly in the next step without purification. Exact mass m / z calculated for C29H31N03S 473.2, found (LC / MS) 474.3.

Synthesis of (4- (ethylthiocarbonyl)) phenylalanine (1): a solution of ld (0.94 g, 2.0 mmoles) from the previous step in trifluoroacetic acid (8 ml) and CH2C12 (2 ml) was stirred for one hour at room temperature . After the organic solvents were completely removed under reduced pressure, concentrated HCl solution (0.8 ml) and MeOH (10 ml) were added and the resulting solution was stirred for 1 hour at room temperature, after which time, all the solvent was stirring and anhydrous acetone (10 ml) is added. The solution was filtered and the recovered solid was titrated with anhydrous MeOH (2 ml). After filtration, the methanolic filtrate was subjected to reduced pressure to yield 1 as a white solid (> 0.55 g, 95%). XH NMR (400 MHz, DMSO-d6) d 7.85 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 4.21 (t, J = 6.4 Hz, 1H), 3.06 (q, J = 14.8, 17.2 Hz, 2H), 3.00 (s, 2H), 1.26 (t, J = 7.2 Hz, 3H). Exact mass m / z calculated for C? 2H15N03S 253.1, found (LC / MS) 254.2.

GENETIC PROGRAMMING OF INCORPORATION OF P-ETILTIOCARBONIL-L-PHENYLALANIN To genetically encode p-ethylthiocarbonyl-L-phenylalanine in E. coli, it was necessary to generate a pair of orthogonal aminoacyl-tRNA synthetase / orthogonal tRNA specific for this amino acid. Based on the crystal structure, of the complex of TyrRS-tRNA L-tyrosine of M. jannaschii (Kobayashi et al., Nat. Struct. Biol. 2003, 10, 425-432), six residues (Tyr32, Leu65, Phel08, Glnl09, Aspl58 and Leul62) at the TyrRS tyrosine binding site of M. jannaschi i were randomly mutated. A library of 109 TyrRS was passed through three rounds of positive selection (based on the deletion of an amber codon in chloramphenicol acetyltransferase) alternated with two rounds of negative selection (based on the deletion of three amber codons in the barnase gene) in the presence and absence of p-ethylthiocarbonyl-L-phenylalanine, respectively, and a number of clones emerged whose survival in chloramphenicol was dependent on p-ethylthiocarbonyl-L-phenylalanine. It was found that one of these mutants supports cell growth in 120 μg mL of chloramphenicol in the presence of p-ethylthiocarbonyl-L-phenylalanine, and 10 μg mL "1 of chloramphenicol in its absence.

Sequencing of this clone revealed the following mutations: Tyr32Ala, Leu65Phe, PhelOdTrp, Glnl09Ser, Aspl58Ser and Leul62His (see, Table 5, SEQ ID NO: 60). The mutation of Tyr32 to Ala32 probably removes the hydrogen bond between the phenolic hydroxyl group of bound tyrosine and Tyr32.

To confirm that the observed phenotype is caused by the specific incorporation of the p-ethylthiocarbonyl-L-phenylalanine site by the pair of mutRNACuA-mutTyrRS, nn codon amber was replaced by the seventh position (Tyr) in the gene encoding the protein of Z domain (Nilsson et al., Protein Eng. 1987, 1, 107-113) fused to a C-terminal His6 tag. The protein was expressed in the presence or absence of 1 mM p-ethylthiocarbonyl-L-phenylalanine and purified by Ni-NTA chromatography. Analysis by SDS-PAGE showed that the expression of the mutant Z-domain protein was completely dependent on the presence of p-ethylthiocarbonyl-L-phenylalanine. The mutant protein was expressed in approximately 10-30% yield relative to the wild type Z domain protein (~ 8 mg / L in medium minimum containing 1% glycerol, 0.3 mM leucine and 1 mM p-ethylthiocarbonyl-L-phenylalanine with appropriate antibiotics). Additional evidence for the site specific incorporation of p-ethylthiocarbonyl-L-phenylalanine was obtained by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition to the observation of an experimental average mass of 8006 Da for the protein Tyr > p-ethylthiocarbonyl-L-phenylalanine (MT = 8002 Da, see figure 16), a minor peak corresponding to the mutant protein without the first methionine portion in acetylated form (MExperimeneai = 7913 Da vs. MT = 7913 Da) and a peak The highest corresponding to the mutant protein without the first portion of methionine (MExperienai = 7871 Da vs. MToric = 7871 Da) were also detected (figure 16). Another major peak of 7828 Da was present which corresponds to the Z domain protein containing a free carboxylic acid group in place of a thioester portion in position 7, which has a calculated mass of 7827 Da in its protonated form. With the assumption that both acid and thioester-containing mutant proteins have comparable ionization efficiencies under mass detection conditions, the integration of their corresponding peak mass areas suggests that about 40% of the thioester-containing mutant protein is hydrolyzed . The fact that the mutant synthetase does not incorporate the unnatural amino acid p-ethylthiocarbonyl-L-phenylalanine in its hydrolyzed form in vivo and that the p-ethylthiocarbonyl-L-phenylalanine appears to be stable both in vitro and in vivo, suggests that the hydrolysis of the thioester to the acid occurs after its incorporation of the Z-domain protein by the thioester-specific mutant synthetase. To determine whether the thioester side chain of the mutant proteins can be selectively modified, an in vitro chemical ligation was performed with 20-60 μg / of mutant protein containing crude thioester and 10 mM cysteine ethylester in a pH-regulated solution with phosphate containing 100 mM dithiothreitol (DTT) and 2 M guanidinium chloride at a pH of 8.0. Then the resulting modified protein was purified and analyzed by MALDI-TOF MS. The experimental average masses of 7956 Da and 7998 Da, corresponding to thioester-containing proteins modified with a cysteine molecule (MT = 7958 Da and 8000 Da for proteins without the first methionine portion and without the first methionine portion in acetylated form , respectively), were obtained (figure 17). The efficiency of marking could be estimated qualitatively to be greater than 85% by integrating its peak areas of masses. As shown in Figure 16, Z domain proteins are predominantly expressed without the first methionine residue. In this form, the mutant protein containing unmodified thioester has a molecular weight of 7872, which can be superimposed with the acid-containing proteins in acetylated form (MTreor = 7869 Da). Therefore, in the calculation of the marking efficiency, the peak area of 7867 Da was taken as the upper limit value for the unmodified thioester containing proteins, leading to an estimated value greater than 85% of marking efficiency. The peaks at 7825 Da and 7867 Da (MTedrico = 7827 Da and 7869 Da) are indicators of the presence of Z domain proteins that contain a carboxylic acid group in position 7, which is not reactive towards the cysteine ethyl ester. As expected, no labeling product was detected for ZT WT domain proteins, indicating that the labeling reaction occurred only between the cysteine molecule and the thioester group but not any functional group existing in the WT protein. On the other hand, neither intramolecular side chain cyclization nor self-dimerization involving the thioester group and any e-amino group of the five lysine residues in thioester-containing mutant proteins were observed. Accordingly, these data demonstrate the excellent selectivity and reactivity of the thioester handle for reliable and selective in vitro protein modification.

CHEMICAL LIGATION BETWEEN Z-DOMAIN PROTEINS CONTAINING ETHYL ESTER OF CISTEINE AND THIOESTER E. coli DH108 cells (60 ml) harboring the plasmid encoding the mutant tRNA synthetase and the expression vector pLEIZ encoding the Z domain gene with a codon amber in the seventh position and a His6 COOH-terminal tag were cultured at 37CC, induced for four hours at an OD60o of 0.5 by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and agglomerated. To the agglomerated cells is added 1 L of pH buffer solution (6 M guanidinium chloride, 100 mM sodium phosphate, 200 mM sodium chloride, pH = 8.0). The solution was stirred for 1 hour at room temperature, sonicated for three minutes and centrifuged to remove any cellular debris. To the clear supernatant is added 2 ml of phosphate pH buffer (100 mM sodium phosphate, 200 mM sodium chloride, 0.01 M cysteine ethyl ester, pH = 8.0), 150 dithiothreitol solution (2 M) and 60 mg of 2-sodium mercaptoethenesulfonate (MESNA). The solution mixture was stirred for 12 hours at room temperature. The solution containing modified proteins was exchanged and concentrated in 500 μl of buffer solution (8 M urea, 100 mM sodium phosphate, 10 mM Trizm, pH 8.0). The modified proteins were purified by Ni2 + affinity chromatography according to the manufacturer's protocol (Qiagen, Chatsworth, CA), dialysed against distilled water and analyzed by MALDI-TOF MS.

CONCLUSION In conclusion, we have provided a new orthogonal synthetase derived from tyrosyl tRNA synthetase from M. jannaschi i. When used in conjunction with an M. jannaschii suppressor tARNCuA used in conjunction with an M. jannaschi suppressor tARNCuA, these reagents allow in vivo incorporation of the unnatural amino acid p-ethylthiocarbonyl-L-phenylalanine into polypeptide chains. This work illustrates a biosynthetic protocol for the bacterial production of proteins containing a side chain thioester handle at defined sites. This allows highly selective and efficient chemical ligation of a wide variety of ligands to the reactive group on the amino acid residue of p-ethylthiocarbonyl-L-phenylalanine from the amino acid to a protein.

EXAMPLE 14 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid of Dicetone p- (3-oxobutanoyl) -L-phenylalanine to proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of the non-natural amino acid of dicetone p- (3-oxobutanoyl) -L-phenylalanine (see, figure 1) a proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this non-natural amino acid were isolated that function in an E. coll host cell system. Comparative studies as to the ability of simple monoketone or ß-diketone functional groups to form amymas with butylamma and the stabilities of the thus formed phosphate pH buffer at different pHs demonstrate the easy production of enolmoma formed at from the ß-diketone portion at pH ranging from 6.5 to 10.5, as well as its superior stability towards acid hydrolysis up to pH 3.9. In comparison, at a pH of up to 10.5 under the identical conditions, the monoketone group remains essentially as a free form without any detectable γ-formation. Thus, an unnatural amino acid carrying a portion of β-diketone in its side chain was synthesized and the identification of a pair of tARγ-orthogonal enzyme capable of incorporating this non-natural amino acid was undertaken. The invention provides a successfully evolved mutant synthetase that specifically incorporates this amino acid containing diketone to proteins in vivo with high translation efficiency and fidelity. As more fully described hereinbelow, a hydroxylamine biotin derivative was then selectively coupled to this a group of diketone that was genetically encoded to a Z-domain protein, suggesting that the acetone portion could serve as a powerful chemical handle whose reactivity is orthogonal to normal biological chemistries to bring a variety of properties external to the target proteins. It has previously been shown that when adding new components to the Escheri chia coli or yeast translation machinery, non-canonical amino acids could be specifically incorporated into the site with high efficiency and fidelity of protein translation in vitro or in vivo using orthogonal translation components . This method has been used to genetically incorporate ketone-containing amino acids into proteins, which could subsequently be conjugated to non-peptidic molecules with various biological and / or physical properties (eg, polyethylene glycol, biotin, glycomimetics, etc.) by means of the formation of hydrazone and oxime bonds. Although these hydrazone and oxime linkages are stable to physiological conditions, they are disadvantageous by the requirement of the simultaneous presence of two functional groups that are not found among the twenty common amino acids. If a reactive functional group such as a thioester or β-diketone were to be formed directly to form stable adducts with the e-amino group of Usinas or a-amino groups, intermolecular or intramolecular protein cross-links could be formed. For this end, the genetic coding of the amino acid containing diketone 2 in Escherichia coli is now reported. See, Figure 21. It was contemplated that the conjugate product of an aryl diketone 2 with an aliphatic amine can lead to the formation of imine 3 adducts (see, Figure 21), which can be tautomerized to the corresponding enamines stabilized by a bond of intramolecular hydrogen of six members. This can result in a stable adduct at physiological pH. To experimentally verify this reasoning, we begin by measuring the relative reactivity of a simple model system that includes a series of imine formations between butylamine and the aryl monoketone la and the aryl diketone 2 in buffer solution of 100 mM phosphate at different pH which fluctuate from 6.5 to 10.5. The various adducts (lb and 3a-3d) were analyzed using liquid chromatography mass spectrometry (LC / MS). See, Table 4. This Table describes the results of imine formation between butylamine (10 mM) and either aryl monoketone 1 (1 mM) or 2 (1 mM) in pH buffer of PBS (K (P04) 1 100 mM, 500 mM NaCl) at different pH. The reactions were carried out for one week at room temperature.

TABLE 4 Reaction time of 12 hours at room temperature This analysis showed that a pH of up to 10.5, remains essentially as a free form without any detectable formation of lb. In contrast to a pH of 7.4, 50% of 2 has already been converted to 3 and this percentage is increased to a print value of 75% at pH 10.5. This, taken together with the previous observations, that the keto 2B form and the enol -imine 3b form dominates with respect to the corresponding tautomers in aqueous medium (Iglesias, Curr, Org Chem 2004, 8, 1-24, Patteux et al. al., Org Lett 2003, 5, 3061-3063, Aly, Tetrahedron 2003, 50, 1739-1747, Lopez et al., Tetrahedron: Asymmetry 1998, 9, 3741-3744, Mazzone et al., S. Eur. J. Med. Chem. 1986, 21, 277-284, and Kim and Ryu, Bull, Korean, Chem. Soc. 1992, 13, 184-187), confirming by this that the hydrogen bond induces stabilization does not facilitate significantly the production of 3 (predominantly 3b) when compared to the. To further corroborate that the stabilized intramolecular H bond provides 3 greater stability to the hydrolysis of the simple lb. Ib, LC / MS analyzes were also performed in lb and 3 at a pH that fluctuated from 1.9 to 9.4. As shown in Figure 1, 3 (supposedly 3b) remains essentially intact at physiological pH 7.4 or higher. Only -40% conversion from 3 to 2 occurs at a pH of 3.9 after 4 days at room temperature. A more acid treatment of 3 led to complete removal of the amino group (Figure 18). In sharp contrast but as expected, lb is easily hydrolysed at a pH of 10.5 after stirring overnight (data not shown) SYNTHESIS OF NON-NATURAL AMINOACIDE Encouraged by these findings, it is desired to establish the chemistry to derive an unnatural amino acid p- (3-oxobutanoyl) -L-femlalamine that contains a portion of β-diketone in its side chain. The synthesis strategy (see, figure 19) starts from the p-acetyl- (+) - L-phenylalanine easily accessible by protecting the ammo and acid groups of the fundamental chain by Boc chemistry and stepfication, respectively. The addition of a second carbomlo group was carried out under reaction conditions involving potassium tert-butoxide in a mixed solvent (2: 3 [v / v] methyl acetate: THF). Removal of the Boc group with TFA, followed by alkaline hydrolysis, provided the p- (3- oxobutanoyl) -L-phenylalanine with an overall yield of 40%.

IDENTIFICATION OF ORTHOGONAL TRANSLATION COMPONENTS New orthogonal aminoacyl-tRNA synthetase / tRNA pairs were constructed for the in vivo incorporation of p- (3-oxobutanoyl) -L-phenylalanine into proteins using established protocols. Based on the crystal structure of the TyrRS-RNA (Tyr) complex of M. jannaschi i L-tyrosine (Kobayashi et al., Nat. Struct. Biol. 2003, 10, 425-432), six residues (Tyr32, Leu65 , PhelOd, Glnl09, Aspl58 and Leul62) around the TyrRS tyrosine binding site of M. jannaschi i were randomly mutated. After sequentially passing the generated library of approximately 109 mutants through three rounds of positive selection, alternated with two rounds of negative selection according to the published protocol, a number of clones emerged whose survival in chloramphenicol was dependent on the presence of p- (3-oxobutanoyl) -L-phenylalanine. Two TyrRS mutants were identified using an in vivo analysis based on the suppression of the TAG codon Aspll2 in the CAT gene. These two mutants can support cell culture in chloramphenicol 120 μg L "1 in the presence of p- (3-oxobutanoyl) -L-phenylalanine, and up to 10 μg mL" 1 of chloramphenicol without p- (3-oxobutanoyl) -L- phenylalanine This result suggests that the two synthetases Evolved ones have both a higher activity for p- (3-oxobutanoyl) -L-phenylalanine than for any natural amino acid. The DNA sequencing of these mutants revealed that they converge to the same sequence (see Table 5, SEQ ID NO 61) Both hydrogen bonds between the phenolic hydroxy group of bound tyrosma and Tyr32 and Aspl58 are broken by mutations to Gly. Leu65 is converted to Val65, possibly providing more space to accommodate the extended fundamental chain of the β-diketone. Mutations of the PhelOdThr and Leul62Ser as well as a conserved Glnl09 can thus indicate their involvement in the bond of H to the carbonyl oxygen in the portion of β-diketone. The sequences of these smtetase clones are summarized below.

To confirm that the observed phenotype is caused by the specific incorporation of the p- (3-oxobutane? L) site - Tyr L-fen? Lalan? Na by the pair mutRNACUA-mutTyrRS, an amber codon was introduced instead of the codon for tyrosine in the seventh position in the gene encoding the Z domain protein (? Ilsson et al., Protein Eng. 1987, 1, 107-113) fused to a C-terminal H? S6 tag. The protein was expressed in the presence or absence of 1 mM p- (3-oxobutane? L) -L-femlalamine and purified by? I-? TA chromatography. Analysis by SDS-PAGE revealed unnatural amino acid-dependent protein expression (Figure 20). The volume of mutant protein loaded to the gel is three times the broad-type protein (WT) where the unnatural amino acid containing diketone is replaced with a tyrosine residue, indicated at about 30% efficiency of incorporation of p- (3 - oxobutanoyl) -L-femlalamna compared with tyrosma. More convincing evidence for the unambiguous incorporation of p- (3-oxobutanoyl) -L-phenylalanine was obtained by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition to the observation of an experimental average mass of 7991 Da (MTe? R? Co = 7997 Da) for the intact protein, a higher peak corresponding to the protein without the first portion of methionine (MExper? Mentai = 7867 Da, MTeor? co = 7866 Da) was also detected. The ratio of signal to noise was > 400, suggesting fidelity for the incorporation of p- (3-oxobutane? L) -L-phenylalanine better than 99%, using the pair Tyr de mutRNACUA-mutTyrRS developed, The possibility of using a portion of diketone as a chemical handle for the specific modification of the protein site with external properties was tested by carrying out the in vitro labeling of diketone-containing proteins expressed with hydroxylamine derivative biotma (MW = 331.39, purchased from Molecular Probes). Purified mutant proteins and ZT WT domain proteins were treated with biotin 2 mM hydroxylamine in phosphate buffer at a pH of 4.0 at 25 ° C for 12 hours. After dialysis against water to remove the excess biotin hydroxylamine, the proteins were analyzed by MS MALDI-TOF. The experimental average masses of 8315 Da (MTeópco = 8310 Da, biotma-tagged intact mutant protein), 8182 Da (MTecond = 8179 Da, biotma-tagged mutant protein without the first methiomna residue) and 8225 Da (MTecond = 8221 Da, biotin-tagged mutant protein without the first methionine residue to its acetylated form) were obtained, confirming that the biotin hydroxylamine reacted with the mutant Z domain proteins in a molar ratio of 1: 1. As expected, no labeling product was detected for the WT domain Z proteins, indicating that the labeling reaction occurred only between the hydroxylamine and the diketone group, but not any functional group existing in the WT protein. Taken together with no observation of mutant proteins containing unlabeled diketones in the mass spectrum, these data demonstrate the excellent specificity and high reactivity of the diketone handle for the selective m vitro modification of proteins. The present example demonstrates that the incorporation of a ß-diketone handle to live m protein using an evolved and highly specific orthogonal translation system occurs specifically at the site with high fidelity and efficiency that is comparable to its natural counterpart. stability as the production phase of 3 with respect to a wide pH range, the modulation of protein-protein interactions through the formation of Schiff base between the β-diketone portion and the ammo group of a hsina residue it is highly possible, especially when p- (3-oxobutanoyl) -L-femlalamine is placed in a favorable hydrophobic.

EXAMPLE 15 Orthogonal translation components for the in vivo incorporation of the unnatural amino acid p-isopropylthiocarbonyl-L-phenylalanine to proteins in E. coli The present example describes compositions and methods for the biosthetic incorporation of p-isopropylthiocarbonyl-L-femlalanma (see, Figure 1) to proteins using the host cell translation machinery of E. coll. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this non-natural amino acid were isolated that function in a host cell system of E. coli. This non-natural amino acid finds use as a target for post-translational modifications when they are incorporated into proteins and is also advantageous because the chemically reactive portion on the non-natural amino acid is resistant to the activities of hydrolysis of cellular enzymes. A new orthogonal synthetase was derived from tyrosyl-tAR? M. jannaschii synthetase and is used in conjunction with the tAR? CuA suppressor of M. jannaschii. This new orthogonal pair has no affinity or has very low affinity for any of the common amino acids (that is, they occur stably in nature). The tAR? Derivative orthogonal synthetase selectively charges the TAR? CUA amber suppressor with p-isopropylthiocarbonyl-L-phenylalanine. The tAR? Aminoacylated suppressor (ie, the "loaded" TAR) is used as a substrate by the endogenous E. coli translation apparatus to incorporate p-isopropylthiocarbonyl-L-phenylalanine in response to the amber TAG retention codon (a selector codon) found in a transcript. The orthogonality of this pair of TAR? / Synthetase ensures that neither the TAR? nor does the synthetase react cross-reactive with tAR? of endogenous E. coli or synthetases and that the non-natural amino acid is incorporated only in response to a non-sense amber codon, TAG. We undertook a search for orthogonal synthetases that have the ability to specifically load an orthogonal tRNA with p-isopropylthiocarbonyl-L-phenylalanine. This search used the protocols that have been previously described.

A library of tyrosyl-tRNA-synthetase mutants of M. jannaschi i was generated by mutagenesis of wild-type tyrosyl-tRNA-synthetase from M. jannaschi i, where the mutagenesis consisted of randomizing active site residues predicted on the basis of the crystal structure of other aminoacyl tRNA-synthetase molecules. Following mutagenesis, the mutant synthetase library was passed through multiple rounds of positive and negative selection. This selection produced a synthetase clone that had the ability to load the O-tRNA with p-isopropylthiocarbonyl-L-phenylalanine. That synthetase clone was sequenced and the amino acid sequence was determined (see, Table 5, SEQ ID NO: 62). This mutant synthase shows the following substitutions in relation to the M. j annaschi wild type synthetase sequence: EXAMPLE 16 Orthogonal translation components for the in vivo incorporation of non-natural fluorescent amino acids containing coumarin to proteins in E. coli The present example describes compositions and methods for the biosynthetic incorporation of 7-amino-coumarin alanine and 7-hydroxy-coumarin alanine (see, figure 1) to proteins using the host cell translation machinery of E. coli. New orthogonal synthetase / tRNA pairs derived from M. jannaschii to incorporate this unnatural amino acid were isolated that function in an E. coli host cell system. Fluorescence is one of the most sensitive and useful techniques in molecular biology. The discovery of green fluorescent protein (GFP) has led to a spectacular revelation in cell biology, allowing the study of protein expression, localization, dynamics and interaction in living cells through direct visualization (Lippincott- Schwartz et al., Nat. Rev. Mol. Cell Bio. (2001) 2: 444-456). Nevertheless, protein interaction and dynamics can not be targeted at atomic resolution due to the size of GFP. GFP also requires many transcripts to obtain an appropriate signal and requires a time delay for its folding and maturation of fluorophore. The incorporation of fluorescent amino acids, as opposed to a portion of the whole fluorescent protein, would overcome some of the limitations in the GFP fluorescence system. The specific site incorporation of fluorescent amino acids would introduce minimal perturbation to the host protein, which allows the measurement of fluorescence resonance energy transfer (FRET) with much greater accuracy. (Truong and Iura, Curr Opin Struct. Bio, 2001, 11: 573-578). In addition, the use of a fluorescent amino acid will allow probing the local environment of each amino acid position and would target residues that moderate the interaction with other cellular components by varying the position of the fluorescent amino acid in the protein. This would also be very useful in the study of in vitro protein folding (Lakowicz, J. R. Principies of Fluorescence Spectroscopy Ed. 2; Kluwer Academic / Plenum Publishers: New York, 1999), especially in a single molecule system (Lipman et al., Science 2003, 301: 1233-1235), because a protein molecule normally contains more than one residue of tryptophan and dialing Specific chemistry of proteins with fluorescent probes is extremely difficult. The alanine coumarins shown in figure 1 have been synthesized chemically. A new orthogonal synthetase was derived from tyrosyl tRNA synthetase from M. jannaschi i and is used in conjunction with the suppressor tRNACuA M. jannaschii previously described to incorporate these coumarin amino acids. This new orthogonal pair has no affinity or has very low affinity for any of the common amino acids (that is, it occurs stably in nature). The orthogonal tRNA synthetase selectively charges the amber suppressor tARNCuA with 7-amino-coumarin alanine and 7-hydroxy-coumarin alanine. The aminoacylated suppressor tRNA (that is, the "loaded" tRNA) is used as a substrate by the endogenous E. coli translation apparatus to incorporate 7-amino-coumarin alanine and 7-hydroxycoumarin alanine in response to the amber retention codon TAG (a selector codon) found in a transcript. The orthogonality of this tRNA / synthetase pair ensures neither the tRNA nor the synthetase cross-reacts with endogenous E. coli tRNA or synthetases and that the unnatural amino acid is incorporated only in response to TAG. We undertook a search for orthogonal synthetases that have the ability to specifically load an orthogonal tRNA with 7-amino-coumarin alanine or 7-hydroxycoumarin alanine. This search used protocols that have been previously described. A library of tyrosyl-tRNA-synthetase mutants of M. jannaschii was generated by mutagenesis of wild-type tyrosyl-tRNA-synthetase from M. jannaschi, where the mutagenesis consisted of randomizing six active site residues predicted on the basis of the crystal structure of other aminoacyl-tRNA synthetase molecules. The library has a diversity of approximately 109 species. Following mutagenesis, the mutant synthetase library was passed through multiple rounds of positive and negative selection. This selection produced a synthetase clone that had the ability to load the O-tRNA with 7-amino-coumarin alanine or 7-hydroxy-coumarin alanine. Then that synthetase clone was sequenced and the amino acid sequence was determined (see, Table 5, SEQ ID NO: 63). This mutant synthase shows the following substitutions in relation to the tyrosyl-tRNA synthetase sequence of M. jannaschi i wild type: Y32R, L65A, H70M, D158N and L162T. Additional data have been obtained demonstrating the selective incorporation of coumarin alanine amino acids into proteins in response to a selector codon in an orthogonal translation system comprising the isolated synthetase species. These data include (a) expression studies in which a myoglobin gene having a TAG selector codon in position 4 is expressed only in the presence of the non-natural amino acid; (b) the mutant myoglobin synthesized in the presence of the non-natural amino acid appears as a fluorescent band in a SDS-PAGE gel analysis; and (c) the isolated mutant smtetase has been crystallized and the co-crystal structure of the mutant smtetase in the presence of the non-natural amino acid is fluorescent.

EXAMPLE 17 O-RS and O-tRNA species for the incorporation of non-natural amino acids A variety of O-tRNA species can be used with the present invention and the invention is not limited to the use of any particular O-tRNA. For example, O-tRNA species comprising the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2 find use with the invention. With the teachings provided herein, additional O-tRNA species can be constructed for use with the invention. Similarly, O-RS species are also provided (see, Table 5) for use in protocols for the incorporation of non-natural amino acids, for example an unnatural amino acid selected from p-ethylthiocarbonyl-L-fe lalamine, p- (3-oxobutanoyl ) -L-femlalanma, 1, 5-dans? L-alamna, 7-ammo-coumapna alamine, 7 -hydroxy -coumarin alamine, o-mothbenzyl-sepna, O- (2-n? Trobenc? L) -L- tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine; p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine and p-nitro-L-phenylalanine. Polypeptides of O-RS of the invention include those polypeptides comprising the amino acid sequences given in Table 5, SEQ ID NOS: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59-63. Examples of polynucleotides encoding O-RS or portions thereof are also provided. For example, polynucleotides encoding molecules O-RS of the invention include SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 , 43, 45, 47, 51, 56, 58. However, it is not intended that the polynucleotides of the invention are limited to those provided in Table 5. of course, any polynucleotide encoding an amino acid sequence of O-RS of the invention, for example, SEQ ID NOS: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 , 50, 52-55, 57 and 59-63, is also an aspect of the invention. It will be understood that the examples, and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons experienced in the art and will be included in the spirit and scope of this art. application and scope of the appended claims. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to the skilled in the art from this disclosure that various changes in form and details may be made without departing from the true scope of the invention .

For example, all the techniques and apparatuses described above can be used in various combinations. All publications, patents, patent applications and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each publication, patent, patent application and / or other individual document were individually indicated to be incorporated by reference for all purposes.

EXAMPLE 18 Nucleotide and amino acid sequences This example provides nucleotide and amino acid sequences for various polynucleotides and polypeptides, respectively. The sequences provided in Table 5 below are intended to provide examples only and it is not intended that the invention be limited in any way by the sequences provided in Table 5.

TABLES Sequences of nucleotides and amino acids SEQ ID Description SEQUENCE NO: tirusil-tRNAc? A upi-esor e? íeíhon c cc us ja nasc hii CCGGCGGUAGUUCAGCAGGGCAGAACGGCGGACUCUAAAUCCG CAUGGCGCUGGUUCAAAUCCGGCCCGCCGGACCA mutRNA CUA tRNA ^ A GCCCGGAUGGUGGAAUCGGUAGACACAAGGGAUUCUAAAUCCC supre.ssor C culi UCGGCGUUCGCGCUGUGCGGGUUCAAGUCCCGCUCCGGGUACC A MDEFEMIKRNTSEIISEEE REVLKKDEKGAYIGFEPSGK? IL sequence of uminoacids of GHYLQIKKMJ DLQNACFUJ 11 LLADLHAYL.NQKGELÜEI X LO tyrosyl-tARN synthetnsfl of DYNKKVFEA GLKAKYVYGSEFQLDKDYTLNVYRLA KTTLKR? leihan coccus jannaschii ARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGM? QRKTHM ARE PKKVVCIHNPVLTGL.DGEGKMSSS G FIAV (MjTyrRS) wildtype DDSPEEIRAKTKKAYCPAGVVEGNPIMEIAKYF EYP TIKRP E FGGDLTVNSYEELES FKNKE HPMDI.KNAVAEELI ILEP IRKR ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAG CGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTT ACATAGGTTl'TGA? CEC? OTGGTAAA? TACAT'rT? GGGCATTArCTC CAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAAT TATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGT TGGATG? GATTAGAAAAATAGGAGATTATAACAAAAA? GTTTTTGA? GCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCT TGATAAGGATTATACACTGA? TGTCTATAGATTGGCTTTAAAAACTA amino acid sequence of CCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGAT IÍGUMI-I? RIN of GAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTA? Melhanococciis jannaschii TGATATTCATTATTTAGGCGTTGATGTTGCAGTTGGAGGGATGGAGC AGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTT (MjTyrRS) wild type GtTTGT? TTCAC ?? CCCTGTCTTA? CGGGTTTCG? tGCACAACC? 7- GATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAG AAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTT GTtGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATA TCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAG TTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTG CATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGAT TTTAGAGCCAATTAGAAAGAGATTA PY MQEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSM PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPA EGAAVK NTAPAPWTYDNIAYMKNQLKMLGFGYDWSRE? TCT PEYYRWEQKFFTELYKKGLVYKKT? AVNWCPNDQTVLANEQVI DGCCWRCDTKVERKEIPQWFIKITAYADE 1.NDLDK DHWPDT VKTMQRNWIGRSEGVEITFNVND DNTL.TVYTTRPDTFMGCTY LAVAAGHPI? QKAAENNPELAAFIDECRNTKVAEAEMATHE K amino acid sequence of GVDTGFKAVHP TGEEIPVWAANFVLMEYGTGAVMAVPGHDQR Icucil-t? RN .sintetasa (Ec eurs) DYEFASKYGLNIKPVILAADGSEPD SQQALTEKGV FNSGEF wildtype NGLDHEAAFNAIADKLTAMGVGERKVNYRLRD GVSRQRYWGA PIPMVTLEDGTVMFTPDDQLPVILPEDVVMDGITSPIKADPE AKTTVNGMPA RETDTFDTFMESSWYYARYTCPQYKEGM1 .DSF. AANYWLPVDIYIGGIEHAIMHLLYFRFFHK MRDAG VNSDEP AKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIV K? KDAAGHELVYTGMSKMS SKNNGIDPQVMVERYGAOrVR F MHFASP? DMT E QESGVEGANRFLKRV LVYEHTAKGDVAA LNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTA1AAIME LMNKLAKAPTDGEQDRA MQEAL AVVRMLN PFTPliICFTLWQ ELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVP VDATEEQVRERAGQEH VAKYLDGVTVRKVIYVPGKI. N VVG ATGCAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC AGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA CGAGAGCAAAGAGAAGTATTACTGCCTGTCTATGCTTCCCT? T CCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCA TCGGTGACGTGATCGCCCGCTACCAGCATATGCTGGGCAAAAA CGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCG GAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGA CGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCT GGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACG CCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGT ATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTG GTGCCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATC GACGGCTGC? GCTGGCGCTGCGATACCAAAGTTGAACGTAAAG AGATCCCGCAGTGGTTTATCAAAATCAATGCTTACGCTGACGA GCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACC GTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCG TGG? GATCACCTTCAACGTTAACGACTATGACAACACGCTGAC CGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTAC CTGGCGGTACGTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGG AAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAA CACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAA GGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCG ?? G? AATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTA CGGCACGGGCGCAGTTATGGCGGTACCGGGGCACCACCAGCGC GACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGG TTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCA AGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTC AACGGTCTTGACC? TGAAGCGGCCTTCAACGCCATCGCCGATA AACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCG CCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCG CCGATTCCG? TGGTGACGCTGGAAGACGGTACCGTAATGCCGA nucleútiU sequence? S (le CCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGT you? Cil-t? RN sinfetasa (EcLen S) AATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGG wild type GCGAAAACTACCGTTAACGGTATGCCAGCACTGCG? GAAACCG ACACTTTCGACACCTTTATGGAGTCCTCCTGGTACTATGCGCG CTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAA GCGGCTAACTACTGGCTGCCGGTGGATATCTACATTGGTGGTA TTGAACACGCCATTATGCACCTGCTCTACTTCCGCTTCTTCCA CAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCA GCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCT TCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCC GGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTG AAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCA TGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCA GGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTT ATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGG AATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTG GAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCA CTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTC GCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGG CCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAG CTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGG ACCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTAT GCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAG GAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGG TTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGT GGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCG GTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGG TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSAQIGFEPSGKIHL amino acid sequence of tRNA synthetase GHYLQIKKMIDLQNAGFDIIIELADLHAYLNQKGELDEIRKIG aminoacyl-3-a? Or ?? o-L-tyrosine (derived from tyrosyl tRNA synthetase DYNKKVFEAMGLKAKYVYGSEGLLDKDYTLNVYRLALKTTLKR ARRS ELIAREDENPKVAEVI PIMQVNSIHYTGVDVAVGGME Mcthanococcus jannaschii QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV wild type) DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILE? IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTCAGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGAGTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAAGGTTTGCTTGATAAGGATTATAC ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA nucleotide sequence of GCAAGAAG3AGTATGGA? CTTAT? GCAAGAGAGGATGA ?? tRNA synthetase ATC apiinoacil-CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTC 3-amino-tyrosine TATTCATTATACTGGCGTTGATGTTGCAGTTGGAGGGñTGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAACGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA amino acid sequence of clone MDEFEMIKRNTS? IISEE? LREVLKKDEKSAAIGFEPSGKÍHL # 1 of aminoacyl tRNA synthetase-GHYLQIKKMIDLQNAGFDIIIS ADLHAY GELDEIRKIG NQ p-carboxymethyl -fenila the DYNKKVFEAMGLKAKYVYGSERNLDKDYTLNVYRLALKTTLKR girl (derived from tyrosyl tRNA sinteARRSMELIAREDENPKVAEVIYPIMQV Methanococcus jannaschii SIHYHGVDVAVGGME rate QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSS GNFIAV wild tino) DDSPEEIRAKIKKAYCP? G VEGNPIMEIAKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTGCGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATATCGTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA nucleotide sequence of clone # 1 GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA aminoacyl-tRNA synthetase AATATGTTTATGGAAGTGAACGTAATCTTGATAAGGATTATAC p-carboxymethyl-L-fcnilalanina ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAA? GA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTC TATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHL amino acid sequence of clone GHYLQIKKMIDLQNAGFD? IALADLHAYLNQKGELDEIRKIG # 2 of aminoacyl-tRNA sintetasn DYNKKVFEAMGLKAKYVYGSENYLDKDYTLNVYRLALKTT KR p-carboxymethyl-L-fcnilalanina ARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME (tyrosyl tRNA-derived sinteQRKTKMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV Methanococcus jannaschii rate DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTTKRP wildtype) EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAG? GTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTTCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGCTTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGG? GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAAAATTATCTTGATAAGGATTATAC ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA nucleotide sequence of clone # 2 and GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC-tRNA synthetase to inoacil CAAAGGTTGCTGAAGTTATCTATCCAAT ATGC GGTT ATGG >p-curl?; or? i? N-ethyl-L-FCN? lulnnina T? TTC? TT? T ?? GGGCGTTG? TGTTGCAGTTGGAGGGATGG? G CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA? GGTTGTTTGTATTCACAACCCTGTCTT? ACGGGTTTGGATr-G AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGG TCC ??? ?? TA? TGGA? T AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCA? TGG? T? T AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMlKRNTSr.IISEEELREVLKKDEKSASI FEPSGKIHL, ecuencia amino acid GHYLQIKKMIDLUNAGFDIIIALADLHAYLNQKGELDEIRKIG clone # 3 of aminoacyl-tRNA synthetase in DYNKKVFEAMGLKAKYVYGSERQLDKDYTLNVYRLALKTTLKR p-carbo? ETII-L-lcnilalani ?? to ARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME (derived from tyrosyl t RN jannaschii synthetase Methanococcui QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV sihestre type) DDSPEEIRAKIKKAYCPAG VEGNPIMEIAKYFLEYPLTtKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTTCGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGCGTTGGCTGATTTACACGCCTA TTTAAACCAGA? AGGAGAGTTCGATGAGATTAGAAAA? TAGG? GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA sequence of clone nucleotides AATATGTTTATGGAAGTGAACGTCAGCTTGATAAGGATTATAC # "Will ap? ?? or" easy-tRNA .sintetasa ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA p-cnrboximctil-L-phenylalanine GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGG TATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTAT? GCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHL amino acid sequence of clone # 4 GHYLQIKKMIDLQNAGFDIIIALADLHAYLNQKGELDEIRKIG aminoacyl-tRNA sintelasa DYNKKVFEAMGLKAKYVYGSEAQLDKDYTLNVYRLALKTTLKR p-carboxunetil-L-fcnilalanina (tyrosyl tRNA-derived synthesized ARR? Methanococcus jannaschii MELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME rate QRKIHMLARELLPKKVVCGHNPVLTGLDGEGKMSSSKGNFIAV wildtype) DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP TRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTTCGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGCGTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAA? ATAGGA GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAAGCGCAGCTTGATAAGGATTAT? C ACTGAATGTCTATAGATTGGCTTTAAAAACTACCT? AAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC micleótidos sequence of clone CAAAGGTTGCTG? AGTTATCTATCCAATAATGCAGGTTAATGC # of ainiíi? ACLL-tRNA iutetasa TATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGG3ATGGAG p-carboxymethyl-L- phenylalanine C? Gagaa? AATACAC? TGTTAGCA? GGGAGCTTTTACC? AA ?? AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTCGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT? CTGCCCAGCTGGAGTTGTTGAAGG? A? TCCAATAATGG? G? T ACCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAAT? AGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAA CTTATAAAGATTTTAGAGCCA ATT? GAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHL amino acid sequence of clone # 5 GHYLQIKKMIDLQNAGFDIIIALADLHAYLNQKGELDEIRKIG aminoacyl tRNA without rerasa DYNKKVFEAMGLKAKYVYGSEKHLDKDYTLNVYRLALKTTLKR-p-carboxymethyl-L fcnilalaninu ARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME (lirosil derived tRNA ile sinteQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV IASA Mahanococcw; jannaichii wildtype) DDSPEEIRAKIKK YCPAGVVEGNPIME1AKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL? ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTTCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGCGTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA nucleotide sequence of clone GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA aminoacyl-tRNA TFS sintetasn AATATGTTTATGGAAGTGAAAAGCATCTTGATAAGGATTATAC p-carboxymethyl phenylalanine ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGG TATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCT? AGATA GAAAGCAT ?? ? ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAG l 'AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCe.-' GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGG C-'l TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGA I " 'AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGC ^ - ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSG. amino acid sequence of clone GHYLQIKKMIDLQNAGFDIIIGLADLHAYLNQKGELDSi. # 1 of aminoacyl-tRNA synthetase DYNKKVFEAMGLKAK YGSEEPLDKDYTLNVYRLALKT "bifcnilalanina (derived from RRSMELIAREDE PKVAEVIYPIMQVNCIHYHGVDVAVr tyrosyl tRNA synthetase from Methanococcus jannaschii-QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGH wild type.) DDSPEEIRAKIKKAYCP GVVEGNPIMEIAKYFLEYPLT- EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELI IRKRL? ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGA 'TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGAIf-, ATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAAATAC?'; GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAA. CTGGATTTGATATAATTATAGGGTTGGCTGATTTACACG TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAAT GATTAtAACAAAAAAGTTTTTGAAGCAATGGGGTTAA ?. AATATGTTTATGGAAGTGAAGAGCCGCTTGATAAGGATT ? CTGAATGTCTATAGATTGGCTTTAAAAACTACCTT? .. "clone nucleotide sequence GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGA / > # 1 of aminoatil-tRNA synthetase CAAAGGTTGCTGAAGTTATCTATCC? ATA? TGCAGGT "" "biphenylalanine TATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGG.?. CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACC AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTr ?, 'AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAtj GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAA7- ACTGCCCAGCTGGAGTTGTTGAAGGA /? TCCAAT.A T' 'AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAA' GAAAAATTTGGTGGAGATTTG? CAGTTAATAGCTATG? R. ? AGAGñGTTTATTTAAAAATAAGGAATTGCATCCAAT •. AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAC ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPÍ ^ amino acid sequence of clone GHYLQIKKMIDLQNAGFDiriTLADLSAYLNOKGELDE- # 2 of ainin? Acyl-tARN siatetase DYNKKVFEAMGLK? KYV? GSEFQLDKDYTLNVYRL? I,:,. biphenylalanine (derived from ARRSMELIAREDENPKVAEVIYPIMGVNVIHYHGVDVA í; tyrosyl tRNA-synthetase from QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKG; - ' Melhanococcus annaschii DDSPEEIRAKIKKAYCPAGVVEGNPIM IAKYFLEYP. type silvcsti-e) EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIK IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGA;. TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGAT ATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAAATAC .. GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAA, CTGGATTTGATATAATTATAACTTTGGCTGATTTATCTC- TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAA-. nucleotide sequence of clone # 2 tRNA synthetase amiiioacil-GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAG bifenilnlanina AATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATI ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTA / '/ • GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGA .. CAAAGGTTGCTGAAGTTATCTATCCAATAATGGGTGTTÍ- TATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGA: CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACC / - AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGC?. AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGC- ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFE IKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHL amino acid sequence of clone # 1 GHYLQIKKKIDLQNAGFDIIIGLADLHAYLNQKGELDE1RKIG aminoacyl-tRNA synthetase DYNKKVFEAMGLKAKYVYGSEEPLDKDYTLNVYRLALKTTLKR bifcn H l n in (tyrosyl tRNA derived synthetase ARRSMELIAREDENPKVAEVIYPIMQVNCIHYHGVDVAVGGME Methanococcus jannaschii QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFXAV wild type) DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGGGTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAAGAGCCGCTTGATAAGGATT? TAC ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA nucleotide sequence GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC clone # 1 of aminoacyl-t? RN synthetase CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTG bifenilaianina TATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTC? TCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAG? T AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHL amino acid sequence of clone # 2 GH LQIKKMIDLQNAGFDIIITLADLSAYLNQKGELDEIRKIG ainiíioacil-tRNA sintctasa DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKR biphenylalanine (tyrosyl tRNA derived ARRSMELIAREDENPKVAEVIYPIMGVNVIHYHGVDVAVGGME synthetase QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV Methanococcus jannaschii DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP wild type) EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAACTTTGGCTGATTTATCTGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA nucleotide sequence of clone # 2 of amiii? Acyl tRNA synthetase-GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA bifcnilnlanina AATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATAC ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGGGTGTTAATGT TATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHL amino acid sequence of clone # 5 GHYLQIKKMIDLQNAGFDIIIVLADLHAYLNQKGELDEIRKIG aminoacyl-tRNA synthetase of DYNKKVFEAMGLKAKYVYGSEADLDKDYTLNVYRLALKTTLKR biphenylalanine (tyrosyl tRNA derived ARRSMELIAREDENPKVAEVIYPIMQVNSIHYRGVDVAVGGME-sintelasa of CRKIHMLARELLPKK'VCIHMPVLTGLDGEGKMSSSKC.MFIAV Methanococcus jannaschii DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP wild type) EKFGGDLTVNSYEELESLFKNKELHPMDLK AVAEEL1KILEP IRKRL ATGG? CGAATTTGAAATGATAAAGAGAAACACATCTGA? ATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTGGGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAAGCGGATCTTGATAAGGATTATAC ACTGA? TGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC sequence nuclcótidos clone # 5 of apiinoacil-tRNA siutetasa CAAAGG? TGCTGAAGTTATCTATCCAATAATGCAGGTTAATTC blfenilalanina GATTCATT? TCGTGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTG? TTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGA? GGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GA AATTTGGTGGAGATTTGAC? GTTA? TAGCT? TGAGGAGT TAGAGAGTTTATTTA? A ?? TAAGG? ATTGCATCCA? TGGATTt AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAG AGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGK 1L amino acid sequence of clone GHYLQIKKMIDLQNAGFDIIIVLADLHAYLNQKGELDE1RKIG # 6 nmiiioacM-t? RN DYNKKVFEAMGLKAKYVYG3ERPLDKDYTLNVYRLALKTTLKR biphenylalanine synthetase (derived from ARRSMELIAREDENPKVAEVIYPIMQVNGIHYLGVDVAVGGME tyrosyl tRNA synthetase of Methanococcus jannaschii-QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV wild type) DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTTKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATT A GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTA nucleotide sequence of clone TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA No. 6 aminoacyl-tRNA je .sintetasa GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA biphenylalanine AATATGTTTATGGAAGTGAAAGGCCTCTTGATAAGGATTATAC ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTT? ATGG TATTCATTATCTGGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEE? LREVLKKDEKSAHIGFEPSGKIHL amino acid sequence of clone GHYLQ1KKMIDLQNAGFDIIIHLADLHAYLNQKGELDEIRKIG # 7 aminoacyl-tRNA synthetase DYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYRLALKTTLKR bil'enilalaniua (derived from ARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME tRNA synthetase rirosil QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV Melhanococcus jannaschii wild type) DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLK AVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGA / iATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATTT? GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATACATTTGGCTGATTTACACGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATAC ACTGA? TGTCTATAGATTGGCTTTAAAAACTACCTTAA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC sequence AGA ?? n? Cleótidos CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGG clone # 7 aminoacyl-tRNA synthetase GATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAG bifenilaianina CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACC? AAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAG? T AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSAEIGFEPSGKIHL amino acid sequence of clone # 1 of aminoacyl tRNA synthetase-GHYLQIKKMIDLQNAGFDIIIHLADLHAYLNQKGELDEIRKIG bipiridilalanina (tyrosyl tRNA derived DYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYRLALKTTLKR synthetase ARRSMELIAREDENPKVAEVIYPIMQVNGHHYHGVDVAVGGME To faith thanococc us jannaschii QRKIHMLARELLPKKVVCIHMPVLTGLDGEGKMSSSKGNFIAV wild type) DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA ATCTGCTGAGATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG nucleotide sequence of clone # 1 CTGGATTTGATATAATTATACATTTGGCTGATTTACACGCCTA aminoacyl-tRNA sintetnsa TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGA bipiridilalanina GATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATAC ACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGG TCATCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCAT ACTGCCC? GCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCC TGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKTHL clone amino acid sequence GHYLQIKKMIDLQNAGFDIIIYLADLAñYLNQKGELDEIRKIG # 2 of aminoaryl-tRNA synthetase DY KKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKR bipiridilnlamna (derived from ARRSMELIAREDENPKVA? VIYPIMEVNG HYSGVDVAVGGME tyrosyl tRNA synthetase QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFI? Methanococcus jannaschii V DDSPEEIRAKIKKAYCPAG VEGNPI EIAKYFLEYPLTIKRP wild type) EKFGGDLTVNSYEELESLFKNKELHPMD KNAVAEELIKILEP IRKRL ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA TCAGCGAGG? AGAGTTAAGAGAGGTTTTAAAAAAAGA? GAAAA ATCTGCTGGTATAGGTTTTGAACCAAGTGGTAAAATACATTTA GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG CTGGATTTGATATAATTATATATTTGGCTGATTTAGCTGCCTA TTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGG? GA TATAAC GTTTTTGA.AGC ?????? ?? TGGGGTTAAAGGCAA AATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATAC AC? GAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGA GCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC tie nucle sequence? Tidos CAAAGGTTGCTGAAGTTATCTATCCAATAATGGAGGTTAATGG clone # ile "tRNA synthetase minnacil-bipiridilalanina TTGGCATTATAGTGGCGTTGATGTTGCAGTTGGAGGGATGGAG CAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAA AGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGG AGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTT GATGACTCTCCAGA? G? GATTAGGGCTAAGAT? AAGAAAGCA? ACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGAT AGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCA GAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGT TAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTT AAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTT? G? GCCA ATTAGAAAGAGATTATAA MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSANPY PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPA EGAAVK NTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCT PEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVI DGCC RCDTKVERKEIPQ FIKITAYADELLNDLDKLDHWPDT VKTMQRNWIGRSEGVEITF VNDYDNTLTVYTTRPDTF GCTY LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKK amino acid sequence of clone GVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQR aminoacyl-tRNA B8 1,5-dansilalanina DYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEF synthetase (leucyl-derived tRNA NGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGA .sintetasa PIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPE E. coli wild type) AKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSE AANYWLPVDIGIGGIEHAIMTLLYFRFFHKLMRDAGMVNSDEP AKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIV KAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVERYGADTVRLF MMFASPADMTLE QESGVEGANRFLKRVWKLVYEHTAKGDVAA LNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIM? LMNKLAKAPTDGEQDRALN1QEALLAVVRMLNPFTPHICFTL Q ELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVP VDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG ATGGAAGAGCAATACCGCCCGGAAGAGATAG? ATCCAAAGTAC AGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA CGAGAGCAAAGAGAAGTATTACTGCCTGTCTGCTAATCCCTAT CCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCA TCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAA CGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCG GAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGA CGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCT GGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACG CCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGT ATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTG GTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTT? TC GACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAG AGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGA GCTGCTCAACG? TCTGGATAAACTGGATCACTGGCCAGAC? CC GTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCG TGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGAC CGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCT / C sequence nuclc? Tidos clone CTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAA.AGCGGCGG tie aminoacyl? .siutclasa AAAATAATCCTGAACTGGCGGCCTTTATTGACGAA.TGCCGTA RNA / i 1,5-dansilalanina CACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAA GGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCG AAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTA CGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGC GACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGG TTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCA AGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTC AACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATA AACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCG CCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCG CCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGA CCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGT AATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGG GCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCG ACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCG CTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAA GCGGCTAACTACTGGCTGCCGGTGGATATCGGTATTGGTGGTA TTGAACACGCCATTATGACGCTGCTCTACTTCCGCTTCTTCCA CAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCA GCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCT TCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCC GGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTG AAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCA TGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCA GGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTT ATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGG AATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTG GAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCA CTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTC GCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGC CCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAG CTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGG ATCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGT? T GCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAG GAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGG TTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGT GGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCG GTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGG AACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTA? AGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAA MEEQYRPEEIESKVQLH DEKRTFEVTEDESKEKYYCLSANP? PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIG DAFGLPA EGAAVKKWAPAPWTYDNIAYMKNQLKMLGFGYDV7SRELATCT PEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVI DGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDT VKTMQRNWIGRSEGVEITFNVNDYDNTLTVYTTRPDAFMGCTY LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKK GVDTGFKAVHPLTGEEIPV AANFVLH? YGTGAVMAVPGHDQR amino acid sequence T252A aminoacyl-IARN siutctasa DYEFASKYGLNIKPVILAADGSEPDLSQQALTE GVLFNSGEF 1,5-dunsils? Luipnu (derived from leucyl-tRNA NGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRY GA synthetase PIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEW E. coli wild type) AKTTVNGMPALRETD? FDTFMESCHIYARYTCPOY EGM DSE AANYWLPVDIGIGGIEHAIMTLLYFRFFHKLMRDAGMVNSDEP AKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIV KAKDAAGHELVYTGMSKMSKS NNGIDPQVMVERYGADTVRLF MMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAA LNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAA? ME LMNKLAKAPTDGEQDRALMQEALLAWRMLNPFTPHICFTLWQ ELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVP VDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG ATGGAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC AGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA CGAGAGCAAAGAGAAGTATTACTGCCTGTCTGCTAATCCCTAT CCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCA TCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAA CGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCG GAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGA CGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCT GGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACG CCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGT ATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTG GTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATC GACGGCTGCTGCTGGCGCTGCGATACC? AAGTTGAACGTAAAG AGATCCCGCAGTGGTTTATCA? AATCACTGCTTACGCTGACGA GCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACC GTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCG TGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGAC CGTTTACACTACCCGCCCGGACGCGTTTATGGGTTGTACCTAC CTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGG AAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAA CACCAAAGTTGCCGAAGCTGAAATGGCGACG? TGGAG? AAAAA GGCGTCGATACTGGCTTTAAAGCGGTTCACCC? TTAACGGGCG AAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTA CGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGC GACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGG TTATCCTGGCAGCTGACGGCTCTGAGCCAG? TCTTTCTCAGC? AGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTC AACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATA tie niicleótidos sequence T252A tARNsintetasa AACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCG of aiiiinoacil-1, 5-dansilalanina CCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCG CCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGA CCCCGG? CGACCA.GCTGCCGGTGATCCTGCCGGA? GATGTGGT AATGGACGGCATTACCAGCCCGATTAAAGCAG? TCCGGAGTGG GCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCG ACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCG CTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAA GCGGCTAACTACTGGCTGCCGGTGGATATCGGTATTGGTGGTA TTGAACACGCCATTATGACGCTGCTCTACTTCCGCTTCTTCCA C ??? CTGATGCGTGATGCAGGCATGGTGA? CTCTG? CCA? CC? GCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCT TCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCC GGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTG AAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCA TGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCA GGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTT ATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGG AATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTG GAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCA CTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTC GCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGG CCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAG CTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGG ATCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTAT GCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAG GAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGG TTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGT GGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCG GTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGG AACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAA AGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAAGCGGCC MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSANPY PSGRLHMGHVRNYTIGDVTARYQRMLGKNVLQPIGWDAFGLPA EGAAVKNNTAPAPWTYDNIAYMKNQLKMLGE'GYDWSRELATCT PEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQV? DGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDT VKTMQRN IGRSEGVEITFNVNDYDNTLTVYTTRPDTFMGCTY LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKK amino acid sequence V338A GVDTGFKAVHPLTGEEIPV AANFVLMEYGTGAVMAAPGHDQR of tRNA synthetase to inoacil-DYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEF 15-dnnsilalanina (GA derived NGLDHEAAF AIADKLTAMGVGERKVNYRLRDWGVSRQRY tRNA synthetase leucyl PIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEW E. coli wild type) AKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGKLDSE AANYWLPVDIGIGGIEHAIMTLLYFRFFHKLMRDAGMVNSDEP AKQLLCOGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIV KAKDAAGHELVYTGMSKMSKSKNL-JGIDPQVMVERYGADTVRLF MMFASPADMTLEWQESGVEGANRFLKRV KLVYEHTAKGDVAA LNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTATAA1ME LMMKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPIÍICFTLWQ ELKGEGDIDNAP PVADEKAMVEDSTLVVVQVNGKVRAKITVP VDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG ATGGA? GAGCAATACCGCCCGGAAGAGAT? GAATCC? A? GTAC AGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA CGAG? GCAAAGAGAAGTATTACTGCCTGTCTGCTAATCCCT? T CCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCA TCGGTGACGTGATCGCCCGCTACC? GCGTATGCTGGGCAAAAA CGTCCTGCAGCCG? TCGGCTGGGACGCGTTTGGTCTGCCTGCG GAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGA CGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCT GGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACG CCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGT ATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAAC7G GTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATC GACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAG AGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGA GCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACC GTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCG sequence nuclcótidos V338A TGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGAC tRNA synthetase aminoacyl-1,5-dansilalanina CGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTAC CTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGG AAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAA CACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAA AAA GGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCG AAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTA CGGCACGGGCGCAGTTATGGCGGCGCCGGGGCACGACCAGCGC GACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGG TTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGC? AGCCCTGACTGAAAA? GGCGTGCTGTTCAACTCTGGCGAGTTC AACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATA AACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCG CCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCG CCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGA CCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGT AATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGG GCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGA? ACCG ACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCG CTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCG? A GCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACC GTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCG TGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGAC CGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTAC CTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGG AAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAA CACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAA GGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCG AAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTA CGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGC GACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGG TTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCA AGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTC AACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATA AACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCG CCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCG CCGATTCCGATGGTGACGCTGGA? GACGGTACCGTAATGCCGA CCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGT AATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGG GCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCG ACACTTTCG? CACCTTTATGG? GTCCTGCTGGATTTATGCGCG CTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAA GCGGCTAACTACTGGCTGCCGGTGGATATCGCGATTGGTGGTA TTGAACACGCCATTATGGGGCTGCTCTACTTCCGCTTCTTCCA CAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACC? GCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCT TCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCC GGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTG AAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCA TGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCA GGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTT ? TGATGTTTGCTTCTCCGGCTGATATGACTCTCG? ATGGC? GG AATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTG GAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCA CTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTC GCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGG CCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAG CTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGG ACCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTAT GCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAG GAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGG TTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGT GGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCG GTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGG AACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAA AGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAA SPY MEEQYRPEEIESKVQLHWDEKRTFEVTEDEGKEKYYCLS PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIG DAFGLPA EGAAVKNNTAPAP TYDNIAYMKNQLKMLGFGYDWSRELATCT amino acid sequence of clone PEYYR EQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVI aminoacyl-tRNA G2-6 of synthesized DGCCWRCDTKVERKEIPQ FIKITAYADELLNDLDKLDHWPDT TnsA o-nltrobencllscrina (deriVKTMQRNWIGRSEGVEITFNVNDYDNTLTVYASRPDTFMGCT vate tRNA synthetase leucyl-LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKK E. coli wild type) GVDTGFKAVHPLTGEEIPV AANFVLMEYGTGAVMAVPGHDQR DYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLF SGEF NGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRY GA PIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIK? DPEW AKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSe AANYWLPVDIAIGGIEHAIMGLLYFRFFHKLMRDAGMVNSDEP AKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIV KAKDAAGHELVYTGISKMSKSKNNGIDPQVMVERYGADTVRLF MMFASPADMTLEWQESGVEGANRFLKRA KLVYEHTAKGDVAA LNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIME LMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQ ELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVP VDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG ATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGC? C ACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTT ACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAA GT? TAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCA TTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAAT ATTTCAAGCTATACCAAGCATACAATCAACTGAATTCAGTATG GAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTACAGC TTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGACGA GGGCAAAGAGAAGTATTACTGCCTGTCTTGGTCGCCCTATCCT TCTGGTCGACTACACATGGGCCACGTACGTAACTACACCATCG GTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAACGT CCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGG? A GGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGT ACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGG CTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGT &CGCCG GAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTAT? ? A? AAGGCCTGGTATAT? AGA? GACTTCTGCGGTCA? CTGGTG TCCGAACGACCAGACCGTACTGGCGAACGAACAAGTT? TCGAC GGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGA TCCCGC? GTGGTTTATCAAA? TCACTGCTTACGCTGACGAGCT GCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTT AAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGG AGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGT TTACGCTTCCCGCCCGG? CACCTTTATGGGTTGTACCTACCTG clone nucleotide sequence GCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGGAAA G2-? of aminoacyl-tRNA SinteATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACAC rate o-Riiti benzyl serine CAAAGTTGCCGAAGCTGAAATGGCGACGATGGAG? A? A ?? GGC GTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAG AAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGG CACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGCGAC TACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTA TCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGC CCTGACTGAA? AAGGCGTGCTGTTCAACTCTGGCGAGTTC? AC GGTCTTG? CCATGAAGCGGCCTTCA? CGCCATCGCCGATA ?? C TGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCT GCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCG ATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGACCC CGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAAT GGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCG AAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACA CTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCGCTA CACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCG GCTAACTACTGGCTGCCGGTGGATATCGCGATTGGTGGTATTG AACACGCCATTATGGGGCTGCTCTACTTCCGCTTCTTCCACAA ACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCG AAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCT ACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGT TGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAA GCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATAA GC? AAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGT GATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATG

Claims (3)

1. CLAIMS 1. A translation system characterized in that it comprises: (a) a first non-natural amino acid selected from the group consisting of: p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, 1, 5 dansyl-alanine, 7-amino-coumarin-alanine, 7-hydroxy-coumarin-alanine, o-nitrobenzyl-serine, 0- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, m-cyano- L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine and p-isopropylthiocarbonyl-L-phenylalanine; (b) a first orthogonal aminoacyl-tRNA synthetase (O-RS); and (c) a first orthogonal tRNA (O-tRNA); wherein the first O-RS preferably aminoacylates the first O-tRNA with the first non-natural amino acid. 2. The translation system according to claim 1, characterized in that the first O-RS is derived from an aminoacyl-tRNA synthetase of Methanococcus jannaschii. 3. The translation system according to claim 1, characterized in that the first O-RS is derived from a tyrosyl-tRNA synthetase of wild type Methanococcus jannaschii. 4. The translation system in accordance with the claim 1, characterized in that the first O-RS is derived from an ammoacyl-tRNA synthetase from E. coli. 5. The translation system according to claim 1, characterized in that the first O-RS is derived from a leucyl-tAR? E. coll wild type synthetase. 6. The translation system according to claim 1, characterized in that the first O-RS comprises an amino acid sequence selected from the amino acid sequences summarized in SEQ ID? Os: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-54, 57, 59-63 and conservative variants thereof. 7. The translation system according to claim 1, characterized in that the first O-tAR? Is it a TAR? Amber suppressor. 8. The translation system according to claim 1, characterized in that the first O-tAR? comprises or is encoded by a sequence of polynucleotides summarized in SEQ ID? O: 1 or 2. 9. The translation system according to claim 1, characterized in that it further comprises a nucleic acid encoding a protein of interest, the nucleic acid. it comprises at least one selector codon, wherein the selector codon is recognized by the first O-tRNA. 10. The translation system in accordance with the claim 9, characterized in that it further comprises a second O-RS and a second O-tRNA, wherein the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid and wherein the second O-tRNA recognizes a selector codon that is different from the selector codon recognized by the first 0-tRNA. The translation system according to claim 1, characterized in that the system comprises a host cell comprising the first unnatural amino acid, the first O-RS and the first O-tRNA. 12. The translation system according to claim 11, characterized in that the host cell is selected from a eubactepana cell and a yeast cell. 13. The translation system according to claim 11, characterized in that the eubactepana cell is an E. coll cell. 14. The translation system according to claim 11, characterized in that the host cell comprises a polynucleotide encoding the first O-RS. The translation system according to claim 14, characterized in that the polynucleotide comprises a sequence of polynucleotides summarized in SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51 or 58. 16. The translation system according to claim 11, characterized in that the host cell comprises a polynucleotide encoding the first O-tRNA. 17. A method for production in a protein translation system comprising an unnatural amino acid in a selected position, the method is characterized in that it comprises • (a) providing a translation system comprising: (i) a first unnatural amino acid selected from p-ethylthiocarbonyl-L-femlalamine, p- (3-oxobutane? l) -L-phenylalanine , 1, 5-dans? L -plan, 7-am? No-coumann-alam na, 7-hydroxy-coumaplan-alanine, o-m-benzene-senna, O- (2-nitrobenzyl) -L-tyrosma, p carboxymethyl-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-aminotransferase, bipipdilalanm, p- (2-ammo-lh? drox? et? l) -L-phenylalanine and p- Isopropylthiocarbonyl-L-femlalamine; (n) a first ammoacyl-tRNA orthogonal synthetase (O-RS); (m) a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferably ammoacilates the first O-tRNA with the unnatural amino acid; and (iv) a nucleic acid encoding the protein, wherein the nucleic acid comprises at least one codon selector that is recognized by the first O-tRNA; and (b) incorporating the unnatural amino acid at the selected position in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising the unnatural amino acid in the selected position. 18. The method according to claim 17, characterized in that the provision of a translation system comprises providing a pol- yucleotide that encodes O-RS. 19. The method according to claim 17, characterized in that the provision of a translation system comprises providing an O-RS derived from an aminoacyl-tRNA smtetase from Methanococcus jannaschi i. 20. The method of compliance with the claim 27, characterized in that the provision of a translation system comprises providing an O-RS derived from a tyrosyl-tRNA N-syntheta from wild-type Methanococcus jannaschii. 21. The method according to the claim 17, characterized in that the provision of a translation system comprises providing an O-RS derived from an aminoacyl-tRNA synthetase from E. coll. 22. The method according to claim 17, characterized in that the provision of a system of
2. Translation comprises providing an O-RS derived from a wild-type E. coli leucyl-tRNA synthetase. 23. The method according to claim 17, characterized in that the provision of a translation system comprises providing an O-RS comprising an amino acid sequence selected from the amino acid sequences summarized in SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-54, 57, 59-63 and conservative variants thereof. 24. The method of compliance with the claim 17, characterized in that the provision of a translation system comprises mutation of an amino acid binding cavity of a wild-type aminoacyl-tRNA N-synthetase by site-directed mutagenesis and selecting a resulting O-RS that preferably aminoacyl the O-tRNA with the amino acid not natural 25. The method according to claim 24, characterized in that the selection step comprises positively selecting and negatively selecting the O-RS of a cluster comprising a plurality of aminoacyl-tRNA synthetase molecules resulting next from site-directed mutagenesis. 26. The method according to claim 17, characterized in that the provision of a translation system comprises providing a polynucleotide that encodes the O-tRNA. 27. The method according to claim 17, characterized in that the provision of a translation system comprises providing an O-tRNA that is an amber suppressor tARN. The method according to claim 17, characterized in that the provision of a translation system comprises providing an O-tRNA that comprises or is encoded by a polynucleotide sequence summarized in SEQ ID NO: 1 or 2. 29. The method according to claim 17, characterized in that the provision of a translation system comprises providing a nucleic acid comprising an amber selector codon. 30. The method of compliance with the claim 17, characterized in that the protein comprises a second unnatural amino acid that is different from the first unnatural amino acid and wherein the provision of a translation system further comprises a second o-RS and a second O-tRNA, wherein the second O- RS preferably aminoates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid and wherein the second O-tRNA recognizes a selector codon in the nucleic acid that is different from the selector codon recognized by the first O- tRNA. 31. The method of compliance with the claim 17, characterized in that the provision of a translation system comprises providing a host cell, wherein the host cell comprises the first unnatural amino acid, the first O-RS, the first O-tRNA and the nucleic acid, and wherein the step of incorporation comprises cultivating the host cell. 32. The method of compliance with the claim 31, characterized in that the provision of a host cell comprises providing a eubacterial host cell or a yeast host cell. 33. The method of compliance with the claim 32, characterized in that the provision of a eubacterial host cell comprises providing an E. coli host cell. 34. The method according to claim 31, characterized in that the provision of a host cell comprises providing a host cell comprising a polynucleotide that encodes O-RS. 35. The method according to claim 34, characterized in that the step of providing a host cell comprising a polynucleotide encoding the O-RS comprises providing a host cell comprising a polynucleotide comprising a nucleotide sequence summarized in SEQ ID NOs : 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51 or 58. 36. The method according to the claim 17, characterized in that the provision of a translation system comprises providing a cell extract. 37. A translation system characterized in that it comprises: (a) a first non-natural amino acid selected from
3. 3 - . 3-nitro-L-tyrosine and p-nitro-L-phenylalanine; (b) a first orthogonal aminoacyl-tRNA synthetase (O-RS); and (c) a first orthogonal tRNA (O-tRNA); wherein the first O-RS aminoacylates the first O-tRNA with the first non-natural amino acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising the first unnatural amino acid, the first O- tRNA and an O-RS comprising an amino acid sequence selected from SEQ ID NOs: 7-10. 38. The translation system according to claim 37, characterized in that the first O-RS is derived from an aminoacyl-tRNA synthetase of Methanococcus jannaschii. 39. The translation system according to claim 37, characterized in that the first O-RS is derived from a wild type tyrosyl-tRNA synthetase from Me thanococcus jannaschii. 40. The translation system according to claim 37, characterized in that the first O-RS it comprises an amino acid sequence selected from the amino acid sequences summarized in SEQ ID NOs: 7-10 and conservative variants thereof. 41. The translation system according to claim 37, characterized in that the first O-tRNA is an amber suppressor tARN. 42. The translation system according to claim 37, characterized in that the first O-tRNA comprises or is encoded by a polynucleotide sequence summarized in SEQ ID NO: 1. 43. The translation system according to claim 37, characterized in that it further comprises a nucleic acid encoding a protein of interest, the nucleic acid comprises at least one selector codon, wherein the selector codon is recognized by the first O-tRNA. 44. The translation system according to claim 43, characterized in that it further comprises a second O-RS and a second O-tRNA, wherein the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid and wherein the second O-tRNA recognizes a selector codon which is different from the selector codon recognized by the first 0-tRNA. 45. The translation system according to claim 37, characterized in that the system comprises a host cell wherein the host cell comprises the first unnatural amino acid, the first O-RS and the first 0-tRNA. 46. The translation system according to claim 45, characterized in that the host cell is a eubacterial cell. 47. The translation system according to claim 46, characterized in that the eubacterial cell is an E. coli cell. 48. The translation system according to claim 45, characterized in that the host cell comprises a polynucleotide that encodes the first O-RS. 49. The translation system according to claim 48, characterized in that the polynucleotide comprises a nucleotide sequence summarized in SEQ ID NO: 11. 50. The translation system according to claim 45, characterized in that the host cell comprises a polynucleotide encoding the first O-tRNA. 51. A method for producing in a host cell a protein comprising an unnatural amino acid at a specified position, the method is characterized in that it comprises: (a) providing a host cell comprising: (i) a first unnatural amino acid selected from 3-nitro-L-tyrosine and p-nitro-L-phenylalanine; (ii) a first orthogonal tRNA (O-tRNA); (iii) a first orthogonal aminoacyl-tRNA N synthetase (O-RS), wherein the first O-RS preferably undergoes the first O-tRNA with the unnatural amino acid with an efficiency that is at least 50% of the efficiency observed for the host cell comprising the first non-natural amino acid, the first O-tRNA and an O-RS comprising an amino acid sequence selected from SEQ ID NOs: 7-10; and (iv) a nucleic acid encoding the protein, wherein the nucleic acid comprises at least one selector codon that is recognized by the first O-tRNA; and (b) culturing the host cell; and (c) incorporating the unnatural amino acid at the selected position in the protein during translation of the protein, wherein the selected position in the protein corresponds to the position of the selector codon in the nucleic acid, thereby producing the protein comprising the unnatural amino acid in the selected position. 52. The method according to claim 51, characterized in that the provision of a translation system comprises providing a polynucleotide that encodes O-RS. 53. The method according to claim 51, characterized in that the provision of a translation system comprises providing an O-RS derived from a aminoacyl-tRNA synthetase from Methanococcus jannaschii. 54. The method according to claim 51, characterized in that the provision of a translation system comprises providing an O-RS derived from a tyrosyl-tRNA synthetase of wild type Methanococcus jannaschii. 55. The method according to claim 51, characterized in that the provision of a translation system comprises providing an O-RS comprising an amino acid sequence selected from the amino acid sequences summarized in SEQ ID NOs: 7-10 and conservative variants from the same. 56. The method according to claim 51, characterized in that the provision of a translation system comprises the mutation of an amino acid binding cavity of a wild type aminoacyl-tRNA synthetase by site-directed mutagenesis and selecting a resulting O-RS. which preferably aminoacylates O-tRNA with the non-natural amino acid. 57. The method of compliance with the claim 56, characterized in that the selection step comprises selecting positively and negatively selecting the O-RS of a cluster comprising a plurality of aminoacyl-tRNA synthetase molecules resulting immediately from site-directed mutagenesis. 58. The method according to claim 51, characterized in that the provision of a translation system comprises providing a polynucleotide that encodes the O-tRNA. 59. The method of compliance with the claim 51, characterized in that the provision of a translation system comprises providing an O-tRNA that is an amber suppressor tARN. 60. The method according to claim 51, characterized in that the provision of a translation system comprises providing an O-tRNA that comprises or is encoded by a polynucleotide sequence summarized in SEQ ID NO: 1. 61. The method of compliance with claim 41, characterized in that the provision of a translation system comprises providing a nucleic acid comprising an amber selector codon. 62. The method according to claim 51, characterized in that the protein comprises a second unnatural amino acid that is different from the first unnatural amino acid and wherein the provision of a translation system further comprises a second O-RS and a second O -tRNA, wherein the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid and wherein the second O-tRNA recognizes a selector codon in the nucleic acid that is different from the selector codon recognized by the first O-tRNA. 63. The method according to claim 51, characterized in that the provision of a translation system comprises providing a host cell, wherein the host cell comprises the first unnatural amino acid, the first O-RS, the first O-tRNA and the nucleic acid and wherein the incorporation step comprises cultivating the host cell. The method according to claim 63, characterized in that the provision of a host cell comprises providing a eubacterial host cell to a yeast host cell. 65. The method according to claim 64, characterized in that the provision of a eubacterial host cell comprises providing an E. coli host cell. 66. The method according to claim 63, characterized in that the provision of a host cell comprises providing a host cell comprising a polynucleotide encoding the O-RS. 67. The method according to claim 66, characterized in that the step of providing a host cell comprising a polypeptide encoding the O-RS comprises providing a host cell comprising a polynucleotide comprising a nucleotide sequence summarized in SEQ ID NO: 11. 68. The method according to claim 51, characterized in that the provision of a translation system comprises providing a cell extract. 69. a composition characterized in that it comprises a polypeptide comprising an amino acid sequence summarized in SEQ ID NO: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 , 38, 40, 42, 44, 46, 50, 52-54, 57, 59-63 or a conservative variant thereof, wherein the polypeptide of conservative variant aminoacylates a cognate orthogonal tRNA (O-tRNA) with an amino acid unnatural with an efficiency that is at least 50% of the efficiency observed for a translation system comprising the O-tRNA, the unnatural amino acid, and an aminoacyl-tRNA synthetase comprising an amino acid sequence selected from SEQ ID NOs : 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-54, 57 and 59 -63. 70. A polynucleotide characterized in that it encodes the polypeptide according to claim 69. 71. The polynucleotide according to claim 70, characterized in that the polynucleotide is selected from SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51 and 58. 72. The composition according to claim 69, characterized in that the composition is a cell comprising the polypeptide. 73. A composition characterized in that it comprises a polynucleotide comprising a nucleotide sequence summarized in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 , 41, 43, 45, 47, 51 or 58. 74. A vector characterized in that it comprises a polynucleotide according to claim 73. 75. An expression vector characterized in that it comprises a polynucleotide according to claim 73. 76. An characterized in that it comprises a vector, the vector comprises a polynucleotide according to claim 73.